Biomarkers for Early Detection of Ovarian Cancer

ABSTRACT

Three panels of biomarker proteins that can be used in the diagnosis of early-stage ovarian cancer (OC) are described. The biomarker panels not only permit the distinction of patients with ovarian neoplasia (benign or malignant) from normal subjects, but they also allow the identification of patients with early-stage (stage I/II) ovarian cancer from those patients with benign ovarian tumors or normal individuals. The invention additionally provides methods for detecting and treating various cancers, including cancer of the ovary using OC-related molecules.

This application claims the benefit of U.S. provisional patentapplication Nos. 60/674,489, filed Apr. 25, 2005, and 60/588,007, filedJul. 14, 2004, the entire contents of each of which is incorporatedherein by reference.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to describemore fully the state of the art to which this invention pertains.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to detection and therapy ofcancer. The invention is more specifically related to a novel panel ofbiomarkers and their use in early diagnosis and prognosis of women'scancers, particularly ovarian cancer. Antibodies andantisense/interference nucleotides directed against these targets can beused in vaccines and pharmaceutical compositions for the treatment ofvarious cancers expressing the biomarkers identified herein, as well asin methods of detecting and assessing the malignancy of such cancers.The invention further provides methods for identifying molecules usefulin the treatment and detection of cancer.

BACKGROUND OF THE INVENTION

Of the gynecologic malignancies, ovarian cancer has the highestmortality rate. Ovarian cancer often eludes the clinician because of thelack of early symptoms and signs. Hence, ovarian cancer tends to presentat a late clinical stage in >85% of patients and is often followed bythe emergence and outgrowth of chemotherapy-resistant disease in thesepatients after conventional primary cytoreductive surgery and inductionchemotherapy. The American Cancer Society reported that >23,000 womenwere diagnosed with ovarian cancer in the United States in 2002, and 60%of those diagnosed, ≈14,000, are projected to die of their disease. Morewomen die from ovarian cancer than from all other gynecologicmalignancies combined. However, the 5-year survival rate for patientsdiagnosed with early-stage disease is often >90%, but it is <20% foradvanced-stage disease, underscoring the importance of early detection.

The diagnostic and prognostic tumor biomarkers in use today are notadequate in distinguishing benign from malignant ovarian neoplasia andcannot differentiate among the various histological and clinicallyaggressive forms of ovarian cancer. The most commonly used biomarker forclinical screening and prognosis in patients with ovarian cancer isovarian cancer antigen 125 (CA125). Serum CA125 levels are elevated in≈80% of patients with advanced-stage epithelial ovarian cancer but areincreased in only 50-60% of patients with early-stage disease. SerumCA125 levels may be falsely elevated in women with any i.p. pathologyresulting in irritation of the serosa of the peritoneum or pericardium,uterine fibroids, renal disorders, and normal menses. Moreover, serumCA125 levels do not predict the outcome of cytoreductive surgery inpatients with advanced epithelial ovarian cancer.

Ciphergen Biosystems (Fremont, Calif.) has developed the ProteinChip®high-throughput protein expression technology coupled withsurface-enhanced laser desorption/ionization time-of-flight massspectrometry (SELDI-TOF-MS) to facilitate protein profiling of complexbiological mixtures (U.S. Pat. No. 6,881,586, issued Apr. 19, 2005). InSELDI-TOF-MS analysis, a nitrogen laser desorbs theprotein/energy-absorbing molecule mixture from the array surface,enabling the detection of the proteins captured by the array. Theefficacy of the SELDI-TOF-MS technology for the discovery of cancerprotein markers in serum has recently been demonstrated (Rai, A., etal., Arch Pathol. Lab. Med. 126:1518-1526, 2002; Kozak K R, et al., ProcNad Acad Sci USA. 100(21): 12343-12348, 2003).

There remains a need for improved tools to permit the early detectionand prognosis of cancer, particularly ovarian cancer. There also remainsa need for targets useful in the detection and treatment of cancer.

SUMMARY OF THE INVENTION

The invention meets these needs and others by providing materials andmethods for the treatment and detection of cancer. The present inventionidentifies multiple biomarker proteins that can be used in the diagnosisof early-stage ovarian cancer. The biomarker panels not only permit thedistinction of patients with ovarian neoplasia (benign or malignant)from normal subjects, but they also allow the identification of patientswith early-stage (stage I/II) ovarian cancer from those patients withbenign ovarian tumors or normal individuals. In addition, in a blindtest, the biomarker panels described herein distinguished diseased fromhealthy patients.

Cancer can be detected by analyzing a tissue specimen for the presenceof an OC biomarker panel of expression. In one embodiment, the inventionprovides a method of detecting cancer in a specimen from a subjectcomprising examining the expression profile of at least two OC relatedmolecules in the specimen, wherein the OC related molecules are selectedfrom a first group of OC related molecules that are overexpressed inovarian neoplasia and a second group of OC related molecules that areunderexpressed in ovarian neoplasia. Typically, overexpression of amember of the first group of OC related molecules comprises an increaseof at least about two-fold relative to normal tissue, whileunderexpression of a member of the second group of OC related moleculescomprises a decrease of at least about two-fold. In another embodiment,the method for detecting cancer comprises measuring the amount of one ormore OC molecules in a tissue sample of the subject. Typically, themethod comprises measuring at least three biomarkers in the tissuesample. The method further comprises comparing the measurements of thebiomarkers in the tissue sample to a known profile of the biomarkers innormal tissue. This method can be adapted for screening to distinguishpatients with ovarian neoplasia from normal patients, as well as todistinguish patients having benign versus malignant neoplasia.

In one embodiment, the method for detecting cancer comprises contactinga tissue specimen with a detectable molecule that specifically binds anOC molecule and detecting binding of the detectable molecule. Binding ofthe detectable molecule is indicative of cancer. The method foridentifying a cancer that is malignant comprises contacting a cancerspecimen with a detectable molecule that specifically binds an OCmolecule associated with malignancy and detecting binding of thedetectable molecule. Binding of the detectable molecule is indicative ofcancer that is malignant. Examples of a detectable molecule include anantibody directed against an OC protein or an antisense nucleotide thatspecifically hybridizes to an OC nucleic acid molecule. Typically, thecancer cell is derived from ovary, or any other cancer associated withthe overexpression (or a combination of overexpression andunderexpression) of OC molecules described herein.

Representative OC molecules or biomarkers include, but are not limitedto, hemoglobin (α or β), transferrin (TF), apolipoprotein AI (ApoAI),transthyretin (TTR), α1 antitrypsin (α1-AT) and immunoglobulin G (IgG).In one embodiment, the biomarker is α-hemoglobin and/or β-hemoglobin.Also included are any of the additional biomarkers described herein bym/z, as determined by SELDI-TOF-MS.

The invention provides a method of screening for ovarian neoplasia in asubject. In one embodiment, the method comprises measuring hemoglobin ina tissue sample of the subject, and comparing the measured hemoglobin ofthe tissue sample to a measurement of hemoglobin in normal tissue,wherein a two-fold or greater increase in the measured of hemoglobin ofthe tissue sample compared to the measurement of hemoglobin in normaltissue is indicative of ovarian neoplasia. In one embodiment, the methodfurther comprises measuring at least two biomarkers in the tissuesample, wherein the at least two biomarkers are selected from a firstgroup of biomarkers whose presence or up-regulation is associated withovarian cancer and/or a second group of biomarkers whose absence ordown-regulation is associated with ovarian cancer, wherein the firstgroup of biomarkers consists of:

a protein having an m/z of 1.953 kDa, 2.065 kDa, 2.216 kDa, 2.928 kDa,2.937 kDa, 3.143 kDa, 3.423 kDa, 3.427 kDa, 4.144 kDa, 4.375 kDa, 4.456kDa, 4.629 kDa, 5.064 kDa, 7.550 kDa, 7.657 kDa, 7.756 kDa, 8.117 kDa,10.874 kDa, 16.850 kDa, 18.559 kDa, 18.912 kDa, 18.98 kDa, 19.186 kDa,22.959 kDa, 29.19 kDa, 29.512 kDa, 30.103 kDa, 33.217 kDa, 36.296 kDa,42.401 kDa, 53.11 kDa (α1-AT), 53.531 kDa, 83.689 kDa, or 84.133 kDa;

and wherein the second group of biomarkers consists of: a protein havingan m/z of 6.884 kDa, 6.931 kDa, 12.785 kDa (transthyretin), 13.797 kDa(transthyretin), 20.989 kDa, 27.595 kDa, 27.977 kDa (apolipoprotein AI),40.067 kDa, 54.605 kDa, 78.9 kDa (transferrin), 79.909 kDa, 90.834 kDa,91.878 kDa, 92.935 kDa, 105.778 kDa, or 106.624 kDa (IgG).

The method further comprises comparing the measurements of the at leasttwo biomarkers in the tissue sample to a known profile of the at leasttwo biomarkers in normal tissue. A measurement indicating a two-fold orgreater increase in a member of the first group of biomarkers, or atwo-fold or greater decrease in a member of the second group ofbiomarkers, relative to normal tissue, is indicative of ovarianneoplasia. In one embodiment, the at least two biomarkers comprisetransthyretin (12.9 kDa and/or 13.8 kDa), apolipoprotein Al (27.977 kDa)and/or transferrin (78.9 kDa) of the second group of biomarkers.

The tissue sample can comprise serum, blood, plasma, or other suitabletissue specimen. The measuring typically comprises spectrometry orimmunoassay. The spectrometry is typically surface enhanced laserdesorption/ionization (SELDI) mass spectrometry. A typical immunoassaywould be an enzyme immunoassay, such as ELISA.

In one embodiment, the measuring is directed to a panel of biomarkersthat differentiates between normal tissue and neoplasia. In thisembodiment, the first group of biomarkers consists of proteins having anm/z of 4.144 kDa, 4.456 kDa, 7.756 kDa, 15.074 kDa, 15.85 kDa, 18.912kDa, 22.959 kDa, 30.103 kDa and 53.531 kDa, and the second group ofbiomarkers consists of a protein having an m/z of 12.785 kDa. In anotherembodiment, the measuring is directed to a panel of biomarkers thatrelates to malignant neoplasia. In this embodiment, the first group ofbiomarkers consists of proteins having an m/z of 3.143 kDa, 4.456 kDa,5.064 kDa, 7.756 kDa, 8.117 kDa, 16.85 kDa, and 18.559 kDa, and thesecond group of biomarkers consists of proteins having an m/z of 13.797kDa, 20.989 kDa, 27.977 kDa, 78.715 kDa, 92.935 kDa and 106.624 kDa. Inyet another embodiment, the measuring is directed to a screeningbiomarker panel, wherein the first group of biomarkers consists ofproteins having an m/z of 4.456 kDa, 15.85 kDa, 18.912 kDa, 22.959 kDaand 30.103 kDa.

In a further embodiment, the measuring is of a validation biomarkerpanel I (VBPI) consisting of a first VBPI group of biomarkers thatconsists of a protein having an m/z of 3.143 kDa, and a second VBPIgroup of biomarkers that consists of proteins having an m/z of 13.797kDa, 20.989 kDa, 78.715 kDa and 106.624 kDa. A measurement indicating atwo-fold or greater increase in a member of the first VBPI group ofbiomarkers, or a two-fold or greater decrease in a member of the secondVBPI group of biomarkers, relative to normal tissue, is indicative ofmalignant ovarian neoplasia. In a yet further embodiment, the methodfurther comprises measuring a validation biomarker panel II (VBPII)consisting of a first VBPII group of biomarkers that consists ofproteins having an m/z of 5.064 kDa and 16.85 kDa, and a second VBPIIgroup of biomarkers that consists of proteins having an m/z of 27.977kDa and 92.935 kDa. A measurement indicating a two-fold or greaterincrease in a member of the first VBPII group of biomarkers, or atwo-fold or greater decrease in a member of the second VBPII group ofbiomarkers, relative to normal tissue, is indicative of malignantovarian neoplasia.

The invention additionally provides a method of detecting ovarianneoplasia in a test subject, which method comprises measuring biomarkersconsisting of transthyretin, hemoglobin, ApoAI and transferrin in atissue sample from the test subject; and comparing the amount of thebiomarkers in the tissue sample with the amount of biomarkers observedin a tissue sample from a normal subject. Increased hemoglobin anddecreased transthyretin, ApoAI and transferrin are indicative of ovarianneoplasia in the test subject. This method can be used to detect amucinous ovarian tumor.

Also provided is a method of screening for ovarian neoplasia in asubject that comprises measuring at least three biomarkers in a tissuesample of the subject, wherein the at least three biomarkers areselected from a first group of biomarkers whose presence is associatedwith ovarian cancer and a second group of biomarkers whose absence isassociated with ovarian cancer. The first group of biomarkers consistsof:

a protein having an m/z of 1.953 kDa, 2.065 kDa, 2.216 kDa, 2.928 kDa,2.937 kDa, 3.143 kDa, 3.423 kDa, 3.427 kDa, 4.144 kDa, 4.456 kDa, 4.629kDa, 5.064 kDa, 7.550 kDa, 7.657 kDa, 7.756 kDa, 8.117 kDa, 10.874 kDa,15.074 kDa (hemoglobin A), 15.850 kDa (hemoglobin B), 16.850 kDa, 18.559kDa, 18.912 kDa, 18.98 kDa, 19.186 kDa, 22.959 kDa, 29.19 kDa, 29.512kDa, 33.217 kDa, 36.296 kDa, 42.401 kDa, 53.11 kDa (α1-AT), 53.531 kDa,83.689 kDa, or 84.133 kDa;

and the second group of biomarkers consists of: a protein having an m/zof 6.884 kDa, 6.931 kDa, 20.989 kDa, 27.595 kDa, 40.067 kDa, 54.605 kDa,79.909 kDa, 90.834 kDa, 91.878 kDa, 92.935 kDa, 105.778 kDa, or 106.624kDa (IgG).

The method further comprises comparing the measurements of the at leastthree biomarkers in the tissue sample to a known profile of the at leastthree biomarkers in normal tissue. A measurement indicating a two-foldor greater increase in a member of the first group of biomarkets, or atwo-fold or greater decrease in a member of the second group ofbiomarkers, relative to normal tissue, is indicative of ovarianneoplasia. The method can further comprise measuring at least oneadditional biomarker selected from: proteins having an m/z of 4.375 kDaand 30.103 kDa of the first group of biomarkers; and proteins having anm/z of 12.785 kDa (transthyretin), 13.797 kDa (transthyretin), 27.977kDa (ApoA1) and 78.715 kDa (transferrin) of the second group ofbiomarkers. Any of these methods can further comprise measuring thecancer antigen CA125.

The invention further provides a kit. The kit comprises at least oneagent that binds a biomarker selected from α-hemoglobin, β-hemoglobin,alpha1-antitrypsin (α1-AT) and any combination thereof; and instructionsfor use of the at least one agent for determining status of ovarianneoplasia in a test sample. The kit can further comprise a container forhousing the at least one agent. The kit can also comprise at least oneagent that binds a biomarker selected from transthyretin, ApoAI,transferrin, CA125 and any combination thereof. In some embodiments, thekit further comprises a substrate to which the at least one agent isbound. The agent can be an antibody that specifically binds thebiomarker, and/or a mass spectrometry probe. The status of ovarianneoplasia to be detected by the kit can comprise: absence of neoplasia,benign, low malignant potential (LMP) or malignant neoplasia. Themethods provided by the invention include a method for inhibitingproliferation of cancer cells comprising contacting a cancer cell with amolecule that disrupts the biological activity of an OC molecule.Typically, the biological activity comprises specific binding of OC toan OC antibody or expression of an OC polynucleotide. Other methodsprovided include-a method for treating cancer in a subject byadministering to the subject a molecule that disrupts the biologicalactivity of an OC molecule, a method for detecting cancer, and a methodfor identifying a cancer that is malignant.

In addition, the invention provides ovarian cancer (OC) relatedmolecules, compositions and additional kits comprising OC relatedmolecules, and methods of using OC related molecules for the treatmentand detection of cancer. In one embodiment, the invention provides anexpression vector comprising a nucleic acid molecule that encodes an OCprotein operably linked to an expression control sequence. The nucleicacid molecule may encode the OC protein in a sense or anti-senseorientation, depending on the intended use. Also provided are host cellscontaining such expression vectors, which can be used for the productionof OC related molecules. In some embodiments, the nucleic acid moleculeis labeled with a detectable marker, or provided in a composition with apharmaceutically acceptable carrier.

The invention additionally provides OC polypeptides, includingimmunogenic OC peptides. The OC polypeptide may be provided in a varietyof forms, as appropriate for a particular use, including, for example,in a soluble form, immobilized on a substrate, or in combination with apharmaceutically acceptable carrier. Antibodies directed against such OCpolypeptides are also provided. In some embodiments, the antibody islabeled with a detectable marker, or provided in a composition with apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B show differentially expressed ovarian cancer-associatedserum proteins. (1A) Detection of differentially expressed ovariancancer-associated serum proteins. A representative pseudogel view ofSELDI-TOF-MS analysis of serum samples, processed on a SAX2 chipsurface, shows relative abundance of potential ovarian cancer markers.The six spectral protein profiles at the top represent serum fromhealthy individuals, three benign samples are represented in the middlesection, and the six spectral profiles on the bottom represent serumfrom patients with ovarian cancer. (1B) Representative spectral overlayof serum from a healthy (solid line) vs. diseased (dashed line)individual. The overlay shows that there is a decrease of protein 13.9kDa in serum from ovarian cancer patients, whereas concurrently there isan increase of the proteins 15.1 kDa and 15.9 kDa. Numbers in the massspectra represent the observed mass of the marker in that particularsample.

FIGS. 2A-2B show ROC curves and plot of sensitivity and specificity forthe ovarian cancer screening panel. ROC curve analysis was based on 140patients to compare the diagnostic performance of five neoplasiabiomarkers making up the screening panel (4.4 kDa, 15.9 kDa, 18.9 kDa,23.0 kDa, and 30.1 kDa), which were identified by the SAS multivariateanalysis program. The area under the ROC curve is 0.94 (2A), and theweighted sum of the marker intensities (score/index) is plotted as afunction of sensitivity and specificity (2B).

FIGS. 3A-3D show ROC curves and plot of sensitivity and specificity forthe ovarian cancer validation panels I and II. Five malignant neoplasiabiomarkers making up the validation panel I (3.1 kDa, 13.9 kDa, 21.0kDa, 79.0 kDa, and 106.7 kDa) and four markers representing thevalidation panel II (5.1 kDa, 16.9 kDa, 28.0 kDa, and 93.0 kDa) wereidentified by the SAS multivariate analysis program. The areas under theROC curves are 0.94 (3A) and 0.90 (3C). The weighted sum of the markerintensities (score/index) is plotted as a function of sensitivity andspecificity (3B and 3D).

FIG. 4 illustrates determination of the pI for ovarian cancer markers byanion exchange fractionation. SAX2 SELDI-TOF-MS profiles of anionexchange fractions depict differentially expressed ovarian cancermarkers. After desalting and dealbuminization, the serum proteins wereeluted from Ciphergen anion exchange columns with a series of buffersdecreasing in pH and were analyzed on SAX2 ProteinChips as mentioned inMethods and Materials.

FIG. 5 shows that the SELDI profiles of purified TTR, Hb, ApoAI andsero-TF match with the 12.9, 13.9, 15.1, 15.9, 28 and 79 m/z peaks fromthe human serum. Pure proteins (0.1-1 μg) of TTR, Hb, ApoAI and TF,diluted 1:5 in 9M Urea/2%/CHAPS/50 mM Tris-HCl, pH9.0 and furtherdiluted 1:5 in1× PBS/0.1% Triton X-100, pH7.5, were analyzed on SAX2chips and compared with SELDI-TOF-MS peaks obtained for 12.9, 13.9,15.1, 15.9, 28 and 79 m/z peaks from the human serum. Marker sizes in“bold” with black arrows identify the ovarian cancer serum markersaligning with pure proteins and distinguish them from other identifiedserum proteins (indicated by gray arrows).

FIG. 6 shows that immunodepletion studies validate TTR, Hb and TF as theproteins corresponding to the 13.9, 15.1, 15.9 and 79 kDa peaks.Pre-cleared serum was immunoprecipitated with 5-30 μg of antibody andA/G agarose beads. Post pre-cleared and depleted samples were dilutedwith an equal volume of Chaps/Urea buffer (9M Urea/2% Chaps/50 mMTris-HCl pH 9.0), followed by 0.5 volume of binding buffer (1× PBS/0.1%Triton-X100, pH 7.5), and analyzed on SAX2 chips. SAX2 SELDI-TOF-MSprotein profiles show the successful depletion of specific peaks afterimmunoprecipitation with TTR, Hb, and TF antibody. Marker sizes in“bold” with black arrows identify depleted ovarian cancer serum markersand distinguish them from other identified serum proteins that were notdepleted (indicated by gray arrows).

FIG. 7 shows that TTR, Hb, and TF are differentially expressed in serumfrom ovarian cancer patients as determined by Western analysis. Totalserum protein was determined by Bradford assay (Sigma-Aldrich, St. LouisMo.) using BSA standards and equal protein concentrations were loadedonto SDS-PAGE for TTR, Hb and TF. For the TTR Western, 1 μg total serumprotein was resolved on a 15% gel. TFR primary antibody was used at a1:1000 dilution and the secondary anti-rabbit HRP at 1:4,000. For the HbWestern, 30 μg total serum protein was resolved on a 15% gel. Hb primaryantibody was used at a 1:1,000 dilution and the secondary anti-goat HRPat 1:4,000. For the TF Western, 0.1 μg total serum protein was resolvedon a 4-15% gradient gel. TF primary antibody was used at a 1:2,000dilution and secondary anti-rabbit HRP at 1:4,000. Molecular weightprotein markers correlating to each gel are indicated.

FIGS. 8A-8E show that TTR (8A), Hb (8B), ApoAI (8C), TF (8D) and CA125(8E) levels distinguish normal individuals from individuals with earlystage ovarian tumors as determined by ELISA. Serum samples from 27normal individuals, 11 individuals with ovarian LMP and 19 individualswith early stage (I/II) ovarian cancer, were analyzed by ELISA for TTR,Hb, ApoAI, TF and CA125 as described under Materials and Methods. Plateswere read at 450 nm and analyzed using SoftMax Pro v4.3 LS software(Molecular Devices, Sunnyvale, Calif.).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of biomarker proteinsthat can be used in the diagnosis of early-stage ovarian cancer. Thebiomarkers not only permit the distinction of patients with ovarianneoplasia (benign or malignant) from normal subjects, but they alsoallow the identification and distinction of patients with early-stage(stage I/II) ovarian cancer from those patients with benign ovariantumors or normal individuals.

Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “OC” refers to ovarian cancer.

As used herein, “OC related molecule” or “OC biomarker” refers to anyone, alone or in combination with other, of the novel biomarkers or anovel panel of biomarkers identified herein as associated with ovariancancer. A biomarker is associated with ovarian cancer if its level(amount of molecule present) is up-regulated or down-regulated inneoplastic versus normal tissue or in malignant versus non-malignanttissue. “OC related molecule” includes OC polypeptides, polynucleotidesencoding OC polypeptides, polynucleotides complementary to thoseencoding OC polypeptides, antibodies that specifically recognize andbind OC polypeptides.

As used herein, “OC biomarker pattern of expression” refers to a patternof protein expression substantially similar to that shown in FIG. 1herein as “CANCER”; or the pattern shown in FIG. 7 as “Early Stage” or“Late Stage”. This pattern of expression can be detected by any of themethods described herein.

As used herein, “biological activity of OC” refers to the specificbinding of OC to an OC binding partner, such as an OC receptor orantibody, to the expression of an OC polynucleotide, and to the growthregulatory effects of OC related molecules.

As used here, “m/z” or “m/z ratio” refers to mass-to-charge ratio, asdetermined by the SELDI-mass spectroscopy protocol described in U.S.patent publication number 2005/0059013 (Mar. 17, 2005). The masses forthe biomarkers described herein are considered accurate to within 0.15percent of the specified value. Assigned m/z ratios are based on theidentification of peaks in the spectrum that represent the signalgenerated by an analyte. Although peak selection can be done by eye,software is typically used to automate the detection of peaks(Ciphergen's ProteinChip® software). In general, this software functionsby identifying signals having a signal-to-noise ratio above a selectedthreshold and labeling the mass of the peak at the center of the peaksignal. One version of this software clusters all peaks appearing in thevarious spectra within a defined mass range, and assigns a mass (m/z) toall the peaks that are near the mid-point of the mass (m/z) cluster.

As used herein, “polypeptide” includes proteins, fragments of proteins,and peptides, whether isolated from natural sources, produced byrecombinant techniques or chemically synthesized. Polypeptides of theinvention typically comprise at least about 6 amino acids.

As used herein, “tumor protein” is a protein that is expressed by tumorcells. Proteins that are tumor proteins also react detectably within animmunoassay (such as an ELISA) with antisera from a patient with cancer.

An “immunogenic polypeptide,” as used herein is a portion of a proteinthat is recognized (i.e., specifically bound) by a B-cell and/or T-cellsurface antigen receptor. Such immunogenic polypeptides generallycomprise at least 5 amino acid residues, more preferably at least 10,and still more preferably at least 20 amino acid residues of a proteinassociated with cancer or infectious disease. Certain preferredimmunogenic polypeptides include peptides in which an N-terminal leadersequence and/or transmembrane domain have been deleted. Other preferredimmunogenic polypeptides may contain a small N- and/or C-terminaldeletion (e.g., 1-30 amino acids, preferably 5-15 amino acids), relativeto the mature protein.

As used herein, “vector” means a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

The term “nucleic acid” or “polynucleotide” refers to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogs of natural nucleotides that hybridize to nucleic acids in amanner similar to naturally-occurring nucleotides.

As used herein, “antigen-presenting cell” or “APC” means a cell capableof handling and presenting antigen to a lymphocyte. Examples of APCsinclude, but are not limited to, macrophages, Langerhans-dendriticcells, follicular dendritic cells, B cells, monocytes, fibroblasts andfibrocytes. Dendritic cells are a preferred type of antigen presentingcell. Dendritic cells are found in many non-lymphoid tissues but canmigrate via the afferent lymph or the blood stream to the T-dependentareas of lymphoid organs. In non-lymphoid organs, dendritic cellsinclude Langerhans cells and interstitial dendritic cells. In the lymphand blood, they include afferent lymph veiled cells and blood dendriticcells, respectively. In lymphoid organs, they include lymphoid dendriticcells and interdigitating cells.

As used herein, “modified” to present an epitope refers toantigen-presenting cells (APCs) that have been manipulated to present anepitope by natural or recombinant methods. For example, the APCs can bemodified by exposure to the isolated antigen, alone or as part of amixture, peptide loading, or by genetically modifying the APC to expressa polypeptide that includes one or more epitopes.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with an active ingredient, allows theingredient to retain biological activity and is non-reactive with thesubject's immune system. Examples include, but are not limited to, anyof the standard pharmaceutical carriers such as a phosphate bufferedsaline solution, water, emulsions such as oil/water emulsion, andvarious types of wetting agents. Preferred diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline.

Compositions comprising such carriers are formulated by well knownconventional methods (see, for example, Remingion's PharmaceuticalSciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton,Pa., 1990).

As used herein, “a” or “an” means at least one, unless clearly indicatedotherwise.

Diagnostic Methods

The invention provides a method for detecting cancer in a specimencomprising analyzing the specimen for the presence of an OC molecule orbiomarker. Also provided is a method of screening for cancer in asubject. The cancer of interest is typically ovarian cancer. In oneembodiment, the method comprises contacting a specimen or tissue samplefrom the subject to be screened with a molecule that recognizes andbinds an OC molecule. The molecule can be, for example, an antibodydirected against an OC peptide, or an oligonucleotide probe or antisensemolecule directed against an OC nucleic acid molecule. The specimen canbe from a mammal, such as human, bovine, equine, canine, feline,porcine, and ovine tissue. The subject is preferably a human, typicallya woman who would benefit from knowledge of her ovarian cancer status.The specimen can comprise serum, blood, plasma, vaginal secretions,urine, saliva, tears, a tumor specimen, or other suitable specimen.

Some OC biomarkers are present at increased levels in neoplastic versusnormal tissue samples, or in malignant versus non-malignant tissuesamples, while others are absent or down-regulated in these conditions.The data shown in Example 3 below provides an overview of OC biomarkersand their relative levels observed in normal, neoplastic, malignant andnon-malignant specimens. Depending on the particular assay employed inthe method of the invention, the detecting or screening can bedetermined by presence or absence of the relevant OC biomarker(s), or byup-regulation or down-regulation relative to normal or non-malignantspecimens. Up-regulation or down-regulation can be determined on thebasis of a statistically significant increase or decrease in level ofthe biomarker, or by a two-fold or greater increase or decrease in thelevel of biomarker detected in the sample relative to the normal ornon-malignant sample.

In one embodiment, the method comprises use of an immunoassay, such asan ELISA type assay, that employs an OC antibody to detect the presence(or up-regulation or down-regulation) of OC in a specimen. Other assaysinclude a radioimmune assay (RIA), a Western blot assay, or a slot blotassay. The immunoassay can be used either as a simple detection method,or to measure the amount or level of OC biomarker present. Suchmeasurements can be quantitative, by comparing measured amounts to aknown or control amount of the biomarker, or can be used to comparerelative amounts between different specimens or samples. The antibodycan be immobilized on a substrate. The amount or presence of biomarkeris typically determined via assay directed to a detectable marker orother indication of binding of the antibody to biomarker. Measures canbe based on, for example, detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, birefringenceor refractive index (e.g., surface plasmon resonance, ellipsometry, aresonant mirror method, a grating coupler waveguide method orinterferometry). Optical methods include microscopy (both confocal andnon-confocal), imaging methods and non-imaging methods. Electrochemicalmethods include voltametry and amperometry methods. Radio frequencymethods include multipolar resonance spectroscopy. Methods forperforming these assays are readily known in the art. Other methods canbe used to detect the presence or amount of a biomarker in a sample.Typically, the biomarker is first captured on a substrate. Examples ofsuch methods include, but are not limited to, gas phase ion spectrometrymethods, optical methods, electrochemical methods, atomic forcemicroscopy and radio frequency methods. In one embodiment, the methodcomprises mass spectrometry, such as “surface-enhanced laserdesorption/ionization” or “SELDI”. SELDI refers to a method ofdesorption/ionization gas phase ion spectrometry (e.g., massspectrometry) in which the analyte is captured on the surface of a SELDIprobe that engages the probe interface. In “SELDI-MS,” the gas phase ionspectrometer is a mass spectrometer.

Those skilled in the art will appreciate additional variations suitablefor the method of detecting cancer in tissue through detection of an OCmolecule in a specimen. This method can also be used to monitor OClevels in tissue of a patient undergoing treatment for cancer. Thesuitability of an OC-targeted therapeutic regimen for initial orcontinued treatment can be determined by monitoring OC levels using thismethod.

The invention additionally provides a method for identifying a moleculethat inhibits proliferation of cancer cells. The method comprisescontacting a candidate molecule with an OC molecule and determiningwhether the candidate molecule disrupts the biological activity of theOC molecule. Disruption of the biological activity of the OC molecule isindicative of a molecule that inhibits proliferation of cancer cells.Representative OC molecules include antibodies, proteins andnucleotides.

Kits

For use in the diagnostic and therapeutic applications described herein,kits are also within the scope of the invention. Such kits can comprisea carrier, package or container that is compartmentalized to receive oneor more containers such as vials, tubes, and the like, each of thecontainer(s) comprising one of the separate elements to be used in themethod. For example, the container(s) can comprise a probe that is orcan be detectably labeled. The probe can be an antibody orpolynucleotide specific for an OC protein or an OC gene or message,respectively. Alternatively, the kit can comprise a mass spectrometry(MS) probe. The kit can also include containers containing nucleotide(s)for amplification or silencing of a target nucleic acid sequence, and/ora container comprising a reporter-means, such as a biotin-bindingprotein, e.g., avidin or streptavidin, bound to a detectable label,e.g., an enzymatic, florescent, or radioisotope label. The kit caninclude all or part of the amino acid sequence of the OC, or a nucleicacid molecule that encodes such amino acid sequences.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. In addition, a label can be provided on the container to indicatethat the composition is used for a specific therapeutic ornon-therapeutic application, and can also indicate directions for eitherin vivo or in fits use, such as those described above. Directions and orother information can also be included on an insert which is includedwith the kit.

Polynucleotides of the Invention

The invention provides polynucleotides that encode one or more OCpolypeptides, or a portion or other variant thereof. Preferredpolynucleotides comprise at least 15 consecutive nucleotides, preferablyat least 30 consecutive nucleotides and more preferably at least 45consecutive nucleotides, that encode an OC polypeptide. Polynucleotidesthat are fully complementary to any such sequences are also encompassedby the present invention. Polynucleotides may be single-stranded (codingor antisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules, including siRNA. RNA molecules includeHnRNA molecules, which contain introns and correspond to a DNA moleculein a one-to-one manner, and mRNA molecules, which do not containintrons. Additional coding or non-coding sequences may, but need not, bepresent within a polynucleotide of the present invention, and apolynucleotide may, but need not, be linked to other molecules and/orsupport materials. Portions of such OC polynucleotides can be useful asprimers and probes for the amplification and detection of OC relatedmolecules in tissue specimens.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an OC polypeptide or a portion thereof) or maycomprise a variant of such a sequence. Polynucleotide variants containone or more substitutions, additions, deletions and/or insertions suchthat the immunogenicity of the encoded polypeptide is not diminished,relative to a native OC protein. Variants preferably exhibit at leastabout 70% identity, more preferably at least about 80% identity and mostpreferably at least about 90% identity to a polynucleotide sequence thatencodes a native OC protein or a portion thereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Adas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor.11:105; Santou, N., Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Nad. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e. gaps) of 20 percent or less, usually 5 to 15 percent, or10 to 12 percent, as compared to the reference sequences (which does notcomprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a native OCprotein (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrtolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

Polynucleotides may be prepared using any of a variety of techniquesknown in the art. DNA encoding an OC protein may be obtained from a cDNAlibrary prepared from tissue expressing an OC protein mRNA. Accordingly,human OC DNA can be conveniently obtained from a cDNA library preparedfrom human tissue. The OC protein-encoding gene may also be obtainedfrom a genomic library or by oligonucleotide synthesis. Libraries can bescreened with probes (such as antibodies to OC or oligonucleotides of atleast about 20-80 bases) designed to identify the gene of interest orthe protein encoded by it. Screening the cDNA or genomic library withthe selected probe may be conducted using standard procedures, such asthose described in Sambrook et al., Molecular Cloning: A LaboratoryManual (New York: Cold Spring Harbor Laboratory Press, 1989). Analternative means to isolate the gene encoding OC is to use PCRmethodology (Sambrook et al., supra; Dieffenbach et al., PCR Primer: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

The oligonucleotide sequences selected as probes should be sufficientlylong and sufficiently unambiguous that false positives are minimized.The oligonucleotide is preferably labeled such that it can be detectedupon hybridization to DNA in the library being screened. Methods oflabeling are well known in the art, and include the use of radiolabels,such as ³²P-labeled ATP, biotinylation or enzyme labeling. Hybridizationconditions, including moderate stringency and high stringency, areprovided in Sambrook et al., supra.

Polynucleotide variants may generally be prepared by any method known inthe art, including chemical synthesis by, for example, solid phasephosphoramidite chemical synthesis. Modifications in a polynucleotidesequence may also be introduced using standard mutagenesis techniques,such as oligonucleotide-directed site-specific mutagenesis (see Adelmanet al., DNA 2:183, 1983). Alternatively, RNA molecules may be generatedby in vitro or in vivo transcription of DNA sequences encoding an OCprotein, or portion thereof, provided that the DNA is incorporated intoa vector with a suitable RNA polymerase promoter (such as T7 or SP6).Certain portions may be used to prepare an encoded polypeptide, asdescribed herein. In addition, or alternatively, a portion may beadministered to a patient such that the encoded polypeptide is generatedin vivo (e.g., by transfecting antigen-presenting cells, such asdendritic cells, with a cDNA construct encoding an OC polypeptide, andadministering the transfected cells to the patient).

Any polynucleotide may be further modified to increase stability invivo. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends; the use ofphosphorothioate or 2′ O-methyl rather than phosphodiesterase linkagesin the backbone; and/or the inclusion of nontraditional bases such asinosine, queosine and wybutosine, as well as acetyl- methyl-, thio- andother modified forms of adenine, cytidine, guanine, thymine and uridine.

Nucleotide sequences can be joined to a variety of other nucleotidesequences using established recombinant DNA techniques. For example, apolynucleotide may be cloned into any of a variety of cloning vectors,including plasmids, phagemids, lambda phage derivatives and cosmids.Vectors of particular interest include expression vectors, replicationvectors, probe generation vectors and sequencing vectors. In general, avector will contain an origin of replication functional in at least oneorganism, convenient restriction endonuclease sites and one or moreselectable markers. Other elements will depend upon the desired use, andwill be apparent to those of ordinary skill in the art.

Within certain embodiments, polynucleotides may be formulated so as topermit entry into a cell of a mammal, and to permit expression therein.Such formulations are particularly useful for therapeutic purposes, asdescribed below. Those of ordinary skill in the art will appreciate thatthere are many ways to achieve expression of a polynucleotide in atarget cell, and any suitable method may be employed. For example, apolynucleotide may be incorporated into a viral vector such as, but notlimited to, adenovirus, adeno-associated virus, retrovirus, or vaccrniaor other pox virus (e.g., avian pox virus). Techniques for incorporatingDNA into such vectors are well known to those of ordinary skill in theart. A retroviral vector may additionally transfer or incorporate a genefor a selectable marker (to aid in the identification or selection oftransduced cells) and/or a targeting moiety, such as a gene that encodesa ligand for a receptor on a specific target cell, to render the vectortarget specific. Targeting may also be accomplished using an antibody,by methods known to those of ordinary skill in the art.

Other formulations for therapeutic purposes include colloidal dispersionsystems, such as macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. A preferred colloidal systemfor use as a delivery vehicle in vitro and in vivo is a liposome (i.e.,an artificial membrane vesicle). The preparation and use of such systemsis well known in the art.

Antisense Molecules

The antisense molecules of the present invention comprise a sequencesubstantially complementary, or preferably fully complementary, to allor a fragment of an OC gene. Included are fragments of oligonucleotideswithin the coding sequence of an OC gene.

Antisense oligonucleotides of DNA or RNA complementary to sequences atthe boundary between introns and exons can be employed to prevent thematuration of newly-generated nuclear RNA transcripts of specific genesinto mRNA for transcription. Antisense RNA complimentary to specificgenes can hybridize with the mRNA for that gene and prevent itstranslation. The antisense molecule can be DNA, RNA, or a derivative orhybrid thereof. Examples of such derivative molecules include, but arenot limited to, peptide nucleic acid (PNA) and phosphorothioate-basedmolecules such as deoxyribonucleic guanidine (DNG) or ribonucleicguanidine (RNG).

Antisense RNA can be provided to the cell as “ready-to-use” RNAsynthesized in vitro or as an antisense gene stably transfected intocells which will yield antisense RNA upon transcription. Hybridizationwith mRNA results in degradation of the hybridized molecule by RNAse Hand/or inhibition of the formation of translation complexes. Both resultin a failure to produce the product of the original gene.

Both antisense RNA and DNA molecules and ribozymes of the invention maybe prepared by any method known in the art for the synthesis of RNAmolecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro or in vivotranscription of DNA sequences encoding the antisense RNA molecule. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,antisense cDNA constructs that synthesize antisense RNA constitutivelyor inducibly can be introduced into cell lines, cells or tissues.

DNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences of the 5′ and/or 3′ ends of the moleculeor the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Othermodifications include the use of chimeric antisense compounds. Chimericantisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics. Such compounds havealso been referred to in the art as hybrids or gapmets. RepresentativeUnited States patents that teach the preparation of such hybridstructures include, but are not limited to, U.S. Pat. Nos. 5,700,922 and6,277,603.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Antisense compositions of the invention include oligonucleotides formedof homopyrimidines that can recognize local stretches of homopurines inthe DNA double helix and bind to them in the major groove to form atriple helix. See: Helen, C and Toulme, J. J. Specific regulation ofgene expression by antisense, sense, and antigene nucleic acids.Biochem. Biophys Acta, 1049:99-125, 1990. Formation of the triple helixwould interrupt the ability of the specific gene to undergotranscription by RNA polymerase. Triple helix formation usingmyc-specific oligonucleotides has been observed. See: Cooney, M, et al.Science 241:456-459.

Antisense sequences of DNA or RNA can be delivered to cells. Severalchemical modifications have been developed to prolong the stability andimprove the function of these molecules without interfering with theirability to recognize specific sequences. These include increasing theirresistance to degradation by DNases, including phosphotriesters,methylphosphonates, phosphorothioates, alpha-anomers, increasing theiraffinity for binding partners by covalent linkage to variousintercalating agents such as psoralens, and increasing uptake by cellsby conjugation to various groups including polylysine. These moleculesrecognize specific sequences encoded in mRNA and their hybridizationprevents translation of and increases the degradation of these messages.

Antisense compositions including oligonucleotides, derivatives andanalogs thereof, conjugation protocols, and antisense strategies forinhibition of transcription and translation are generally described in:Antisense Research and Applications, Crooke, S. and B. Lebleu, eds. CRCPress, Inc. Boca Raton Fla. 1993; Nucleic Acids in Chemistry and BiologyBlackburn, G. and M. J. Gait, eds. IRL Press at Oxford University Press,Inc. New York 1990; and Oligonucleotides and Analogues: A PracticalApproach Eckstein, F. ed., IRL Press at Oxford University Press, Inc.New York 1991; which are each hereby incorporated herein by referenceincluding all references cited therein which are hereby incorporatedherein by reference.

RNA Interference

Long double-stranded RNAs (dsRNAs; typically >200 nt) can be used tosilence the expression of a target gene. The dsRNAs enter a cellularpathway that is commonly referred to as the RNA interference (RNAi)pathway. First, the dsRNAs get processed into 20-25 nucleotide (nt)small interfering RNAs (siRNAs) by an RNase III-like enzyme called Dicer(initiation step). Then, the siRNAs assemble intoendoribonuclease-containing complexes known as RNA-induced silencingcomplexes (RISCs), unwinding in the process. The siRNA strandssubsequently guide the RISCs to complementary RNA molecules, where theycleave and destroy the cognate RNA (effecter step). Cleavage of cognateRNA takes place near the middle of the region bound by the siRNA strand.

In mammalian cells, introduction of long dsRNA (>30 nt) initiates apotent antiviral response, exemplified by nonspecific inhibition ofprotein synthesis and RNA degradation. The mammalian antiviral responsecan be bypassed, however, by the introduction or expression of siRNAs(Ambion, Inc., Austin, Tex.).

OC Polypeptides

OC polypeptides as described herein may be of any length. Exemplarylengths include, but are not limited to, up to 10, 15, 20, 25, 30, 40,50, 60, 70, 80, 90 and 100 amino acids or more. Additional sequencesderived from the native protein and/or heterologous sequences may bepresent, and such sequences may, but need not, possess further ligandbinding, immunogenic or antigenic properties. Those skilled in the artwill appreciate that other portions or variants thereof will be usefulin the treatment and detection of cancer.

Immunogenic polypeptides may generally be identified using well knowntechniques, such as those summarized in Paul, Fundamental Immunology,4th ed., 663-665 (Lippincott-Raven Publishers, 1999) and referencescited therein. Such techniques include screening polypeptides for theability to react with antigen-specific antibodies, antisera and/orT-cell lines or clones. As used herein, antisera and antibodies areantigen-specific if they specifically bind to an antigen (i.e., theyreact with the protein in an ELISA or other immunoassay, and do notreact detectably with unrelated proteins). Such antisera and antibodiesmay be prepared using well known techniques. An immunogenic polypeptidecan be a portion of a native protein that reacts with such antiseraand/or T-cells at a level that is not substantially less than thereactivity of the full length polypeptide (e.g., in an ELISA and/orT-cell reactivity assay). Such immunogenic portions may react withinsuch assays at a level that is similar to or greater than the reactivityof the full length polypeptide. Such screens may generally be performedusing methods well known to those of ordinary skill in the art, such asthose described in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. For example, a polypeptide may beimmobilized on a solid support and contacted with patient sera to allowbinding of antibodies within the sera to the immobilized polypeptide.Unbound sera may then be removed and bound antibodies detected using,for example, ¹²⁵I-labeled Protein A.

An OC polypeptide of the invention can comprise a variant of a native OCprotein. A polypeptide “variant,” as used herein, is a polypeptide thatdiffers from a native OC protein in one or more substitutions,deletions, additions and/or insertions, such that the immunogenicity ofthe polypeptide is not substantially diminished. In other words, theability of a variant to react with antigen-specific antisera may beenhanced or unchanged, relative to the native protein, or may bediminished by less than 50%, and preferably less than 20%, relative tothe native protein. Such variants may generally be identified bymodifying one of the above polypeptide sequences and evaluating thereactivity of the modified polypeptide with antigen-specific antibodiesor antisera as described herein. Preferred variants include those inwhich one or more portions, such as an N-terminal leader sequence, havebeen removed. Other preferred variants include variants in which a smallportion (e.g., 1-30 amino acids, preferably 5-15 amino acids) has beenremoved from the N- and/or C-terminal of the mature protein. Polypeptidevariants preferably exhibit at least about 70%, more preferably at leastabout 90% and most preferably at least about 95% identity (determined asdescribed above) to the identified polypeptides.

Preferably, a variant contains conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. Amino acid substitutions may generally be made on the basisof similarity in polarity, charge, solubility, hydrophobicity,hydrophllicity and/or the amphipathic nature of the residues. Forexample, negatively charged amino acids include aspattic acid andglutamic acid; positively charged amino acids include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophllicity values include leucine, isoleucine and valine;glycine and alanine; asparagine and glutamine; and serine, threonine,phenylalanine and tyrosine. Other groups of amino acids that mayrepresent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also,or alternatively, contain nonconservative changes. In a preferredembodiment, variant polypeptides differ from a native sequence bysubstitution, deletion or addition of five amino acids or fewer.Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theimmunogenicity, secondary structure and hydropathic nature of thepolypeptide.

Polypeptides may comprise a signal (or leader) sequence at theN-terminal end of the protein that co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-FEs), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

In some embodiments, the polypeptides are purified from the same subjectto whom the composition will be administered. In these embodiments, itmay be desirable to increase the number of tumor or infected cells. Sucha scale up of cells could be performed in vitro or in vivo, using, forexample, a SCID mouse system. Where the cells are scaled up in thepresence of non-human cells, such as by growing a human subject's tumorin a SCID mouse host, care should be taken to purify the human cellsfrom any non-human (e.g., mouse) cells that may have infiltrated thetumor. In these embodiments in which the composition will beadministered to the same subject from whom the polypeptides arepurified, it may also be desirable purify several OC polypeptides tooptimize the efficacy of a limited quantity of starting material.

Recombinant polypeptides encoded by DNA sequences as described above maybe readily prepared from the DNA sequences using any of a variety ofexpression vectors known to those of ordinary skill in the art.Expression may be achieved in any appropriate host cell that has beentransformed or transfected with an expression vector containing a DNAmolecule that encodes a recombinant polypeptide. Suitable host cellsinclude prokaryotes, yeast and higher eukaryotic cells. Preferably, thehost cells employed are E. coli, yeast, insect cells or a mammalian cellline such as COS or CHO. Supernatants from suitable host/vector systemsthat secrete recombinant protein or polypeptide into culture media maybe first concentrated using a commercially available filter. Followingconcentration, the concentrate may be applied to a suitable purificationmatrix such as an affinity matrix or an ion exchange resin. Finally, oneor more reverse phase HPLC steps can be employed to further purify arecombinant polypeptide.

Portions and other variants having fewer than about 100 amino acids, andgenerally fewer than about 50 amino acids, may also be generated bysynthetic means, using techniques well known to those of ordinary skillin the art. For example, such polypeptides may be synthesized using anyof the commercially available solid-phase techniques, such as theMerrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

Polypeptides can be synthesized on a Perkin Elmer/Applied BiosystemsDivision 430A peptide synthesizer using FMOC chemistry with HPTU(O-BenzotriazoleN,N,N′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence may be attached to the amino terminusof the peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support may be carried out using the followingcleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides may be precipitated in coldmethyl-t-butyl-ether. The peptide pellets may then be dissolved in watercontaining 0.1% trifluoroacetic acid TFA) and lyophilized prior topurification by C18 reverse phase HPLC. A gradient of 0%-60%acetonitrile (containing 0.1% TFA) in water may be used to elute thepeptides. Following lyophilization of the pure fractions, the peptidesmay be characterized using electrospray or other types of massspectrometry and by amino acid analysis.

Fusion Proteins

In some embodiments, the polypeptide is a fusion protein that comprisesmultiple polypeptides as described herein, or that comprises at leastone polypeptide as described herein and an unrelated sequence. In someembodiments, the fusion protein comprises an OC polypeptide and animmunogenic polypeptide. The immunogenic polypeptide can comprise, forexample, all or a portion of an additional tumor protein.

Additional fusion partners can be added. A fusion partner may, forexample, serve as an immunological fusion partner by assisting in theprovision of T helper epitopes, preferably T helper epitopes recognizedby humans. As another example, a fusion partner may serve as anexpression enhancer, assisting in expressing the protein at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the protein or to enable the protein to be targeted todesired intracellular compartments. Still further fusion partnersinclude affinity tags, which facilitate purification of the protein.

Fusion proteins may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion protein isexpressed as a recombinant protein, allowing the production of increasedlevels, relative to a non-fused protein, in an expression system.Briefly, DNA sequences encoding the polypeptide components may beassembled separately, and ligated into an appropriate expression vector.The 3′ end of the DNA sequence encoding one polypeptide component isligated, with or without a peptide linker, to the 5′ end of a DNAsequence encoding the second polypeptide component so that the readingframes of the sequences are in phase. This permits translation into asingle fusion protein that retains the biological activity of bothcomponent polypeptides.

A peptide linker sequence may be employed to separate the first and thesecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion protein usingstandard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea et.al., Gene 40:39-46, 1985; Murphy et al., Proc. Nad. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located 5′ to the DNAsequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals arepresent 3′ to the DNA sequence encoding the second polypeptide.

Fusion proteins are also provided that comprise a polypeptide of thepresent invention together with an unrelated immunogenic protein.Preferably the immunogenic protein is capable of eliciting a memoryresponse. Examples of such proteins include tetanus, tuberculosis andhepatitis proteins (see, for example, Stoute et al., New Engl. J. Med.336:86-91, 1997).

Within preferred embodiments, an immunological fusion partner is derivedfrom protein D, a'surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS I (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292,1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAR This property has beenexploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionprotein. A repeat portion is found in the C-terminal region starting atresidue 178. A particularly preferred repeat portion incorporatesresidues 188-305.

In general, polypeptides (including fusion proteins) and polynucleotidesas described herein are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

Antibodies

The term “antibody” is used in the broadest sense and specificallycovers single anti-OC monoclonal antibodies (including agonist,antagonist and neutralizing antibodies) and anti-OC antibodycompositions with polyepitopic specificity. The term “monoclonalantibody” (mAb) as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e. the antibodiescomprising the individual population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.

The invention provides antibodies that bind to OC proteins andpolypeptides. The most preferred antibodies will specifically bind to anOC protein and will not bind (or will bind weakly) to non-OC proteinsand polypeptides. An antibody “specifically binds” to a protein (orpeptide) if it is capable of a binding reaction with that protein thatis determinative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. For example, the antibodybinds to a particular protein at least two times as much as to thebackground and does not substantially bind in a significant amount toother proteins present in the sample.

Anti-OC antibodies that are particularly contemplated include monoclonaland polyclonal antibodies as well as fragments containing the antigenbinding domain and/or one or more complementarity determining regions ofthese antibodies. As used herein, an antibody fragment is defined as atleast a portion of the variable region of the immunoglobulin moleculethat binds to its target, i.e., the antigen binding region.

OC antibodies of the invention may be particularly useful in cancerdiagnostic and prognostic assays, and imaging methodologies.Intracellularly expressed antibodies (e.g., single chain antibodies) maybe therapeutically useful in treating cancers in which the expression ofOC is involved, such as for example advanced and metastatic braincancers, as well as cancers of the lung, breast, colon or prostate. Alsouseful in therapeutic methods for treatment of cancer are systemicallyadministered OC antibodies that interfere with OC function or thattarget cells expressing OC for delivery of a toxin or therapeuticmolecule. Such delivery of a toxin or therapeutic molecule can beachieved using known methods of conjugating a second molecule to the OCantibody or fragment thereof. Similarly, such antibodies may be usefulin the treatment, diagnosis, and/or prognosis of other cancers, to theextent OC is also expressed or overexpressed in other types of cancer.

The invention also provides various immunological assays useful for thedetection and quantification of OC polypeptides. Such assays generallycomprise one or more OC antibodies capable of recognizing and binding anOC, and may be performed within various immunological assay formats wellknown in the art, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), and the like. Inaddition, immunological imaging methods capable of detecting cancersexpressing OC are also provided by the invention, including but notlimited to radioscintigraphic imaging methods using labeled OCantibodies. Such assays may be clinically useful in the detection,monitoring, and prognosis of OC expressing cancers.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using an OC protein, peptide, or fragment, in isolated orimmunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds.,Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press,NY (1989)). In addition, fusion proteins of OC may also be used, such asan OC GST-fusion protein. In another embodiment, an OC peptide may besynthesized and used as an immunogen.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the OC protein can also be produced in the context ofchimeric or CDR grafted antibodies of multiple species origin. Humanizedor human OC antibodies may also be produced and are preferred for use intherapeutic contexts. Methods for humanizing murine and other non-humanantibodies by substituting one or more of the non-human antibody CDRsfor corresponding human antibody sequences are well known (see forexample, Jones et al., 1986, Nature 321: 522-525; Riechmann et al.,1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239:1534-1536). See also, Carter et al., 1993, Proc. Nad. Acad. Sci. USA 89:4285 and Sims et al., 1993, J. Immunol. 151: 2296. Methods for producingfully human monoclonal antibodies include phage display and transgenicmethods (for review, see Vaughan et al., 1998, Nature Biotechnology 16:535-539).

Fully human OC monoclonal antibodies may be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display) (Griffiths and Hoogenboom, Building an in vitroimmune system: human antibodies from phage display libraries. In:Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic,pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatoriallibraries. Id., pp 65-82). Fully human OC monoclonal antibodies may alsobe produced using transgenic mice engineered to contain humanimmunoglobulin gene loci as described in PCT Patent ApplicationWO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997(see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614).This method avoids the in titro manipulation required with phage displaytechnology and efficiently produces high affinity authentic humanantibodies.

Reactivity of OC antibodies with an OC protein may be established by anumber of well known means, including western blot, immunoprecipitation,ELISA, and FACS analyses using, as appropriate, OC proteins, peptides,OC-expressing cells or extracts thereof.

An OC antibody or fragment thereof of the invention may be labeled witha detectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. A second molecule forconjugation to the OC antibody can be selected in accordance with theintended use. For example, for therapeutic use, the second molecule canbe a toxin or therapeutic agent. Further, bi-specific antibodiesspecific for two or more OC epitopes may be generated using methodsgenerally known in the art. Homodimeric antibodies may also be generatedby cross-linking techniques known in the art (e.g., Wolff et al., CancerRes. 53: 2560-2565).

T Cells

Immunotherapeutic compositions may also, or alternatively, comprise Tcells specific for an OC polypeptide. Such cells may generally beprepared in vitro or ex vivo, using standard procedures. For example, Tcells may be isolated from bone marrow, peripheral blood, or a fractionof bone marrow or peripheral blood of a patient, using a commerciallyavailable cell separation system, such as the ISOLEX™ magnetic cellselection system, available from Nexell Therapeutics, Irvine, Calif.(see also U.S. Pat. No. 5,536,475); or MACS cell separation technologyfrom Miltenyi Biotec, including Pan T Cell Isolation Kit, CD4+ T CellIsolation Kit, and CD8+ T Cell Isolation Kit (see also U.S. Pat. No.5,240,856; U.S. Pat. No. 5,215,926; WO 89/06280; WO 91/16116 and WO92/07243). Alternatively, T cells may be derived from related orunrelated humans, non-human mammals, cell lines or cultures.

T cells may be stimulated with an OC polypeptide, polynucleotideencoding an OC polypeptide and/or an antigen presenting cell (APC) thatexpresses such an OC polypeptide. The stimulation is performed underconditions and for a time sufficient to permit the generation of T cellsthat are specific for the polypeptide. Preferably, an OC polypeptide orpolynucleotide is present within a delivery vehicle, such as amicrosphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for an OC polypeptide if the Tcells kill target cells coated with the polypeptide or expressing a geneencoding the polypeptide. T cell specificity may be evaluated using anyof a variety of standard techniques. For example, within a chromiumrelease assay or proliferation assay, a stimulation index of more thantwo fold increase in lysis and/or proliferation, compared to negativecontrols, indicates T cell specificity. Such assays may be performed,for example, as described in Chen et al., Cancer Res. 54:1065-1070,1994.

Detection of the proliferation of T cells may be accomplished by avariety of known techniques. For example, T cell proliferation can bedetected by measuring an increased rate of DNA synthesis (e.g., bypulse-labeling cultures of T cells with tritiated thymidine andmeasuring the amount of tritiated thymidine incorporated into DNA).Contact with an OC protein (100 ng/ml-100 μg/ml, preferably 200 ng/ml-25μg/ml) for 3-7 days should result in at least a two fold increase inproliferation of the T cells. Contact as described above for 2-3 hoursshould result in activation of the T cells, as measured using standardcytokine assays in which a two fold increase in the level of cytokinerelease (e.g., TNF or IFN-γ) is indicative of T cell activation (seeColigan et al., Current Protocols in Immunology, vol. 1, WileyInterscience (Greene 1998)). T cells that have been activated inresponse to an OC polypeptide, polynucleotide or polypeptide-expressingAPC may be CD4+ and/or CD8+. T cells can be expanded using standardtechniques.

Within preferred embodiments, the T cells are derived from either apatient or a related, or unrelated, donor and are administered to thepatient following stimulation and expansion. For therapeutic purposes,CD4+ or CD8+ T cells that proliferate in response to an OC polypeptide,polynucleotide or APC can be expanded in number either in vitro or invivo. Proliferation of such T cells in vitro may be accomplished in avariety of ways. For example, the T cells can be re-exposed to an OCpolypeptide, with or without the addition of T cell growth factors, suchas interleukin-2, and/or stimulator cells. Alternatively, one or more Tcells that proliferate in the presence of an OC polypeptide can beexpanded in number by cloning. Methods for cloning cells are well knownin the art, and include limiting dilution.

Pharmaceutical Compositions and Vaccines

The invention provides OC polypeptide, polynucleotides, T cells and/orantigen presenting cells that are incorporated into pharmaceuticalcompositions, including immunogenic compositions (i.e., vaccines).Pharmaceutical compositions comprise one or more such compounds and,optionally, a physiologically acceptable carrier. Vaccines may compriseone or more such compounds and an adjuvant that serves as a non-specificimmune response enhancer. The adjuvant may be any substance thatenhances an immune response to an exogenous antigen. Examples ofadjuvants include conventional adjuvants, biodegradable microspheres(e.g., polylactic galactide), immunostimulatory oligonucleotides andliposomes (into which the compound is incorporated; see e.g., Fullerton,U.S. Pat. No. 4,235,877). Vaccine preparation is generally described in,for example, M. F. Powell and M. J. Newman, eds., “Vaccine Design (thesubunit and adjuvant approach),” Plenum Press (NY, 1995). Pharmaceuticalcompositions and vaccines within the scope of the present invention mayalso contain other compounds that may be biologically active orinactive. For example, one or more immunogenic portions of other tumorantigens may be present, either incorporated into a fusion polypeptideor as a separate compound, within the composition or vaccine.

A pharmaceutical composition or vaccine can contain DNA encoding one ormore of the polypeptides as described above, such that the polypeptideis generated in situ. As noted above, the DNA may be present within anyof a variety of delivery systems known to those of ordinary skill in theart, including nucleic acid expression systems, bacteria and viralexpression systems. Numerous gene delivery techniques are well known inthe art, such as those described by Rolland, Crit. Rev. Therap. DrugCarrier Systems 15:143-198, 1998, and references cited therein.Appropriate nucleic acid expression systems contain the necessary DNAsequences for expression in the patient (such as a suitable promoter andterminating signal). Bacterial delivery systems involve theadministration of a bacterium (such as Bacillus-Calmette-Guerrin) thatexpresses an immunogenic portion of the polypeptide on its cell surfaceor secretes such an epitope.

In a preferred embodiment, the DNA may be introduced using a viralexpression system (e.g., vaccinia or other pox virus, retrovirus, oradenovirus), which may involve the use of a non-pathogenic (defective),replication competent virus. Suitable systems are disclosed, forexample, in Fisher-Hoch et al., Proc. Nad. Acad. Sci. USA 86:317-321,1989; Flexner et al., Ann. N. Y. Acad Sci. 569:86-103, 1989; Flexner etal., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330, and5,017,487; WO 89/01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP0,345,242; WO 91/02805; Berkner-Biotechniques 6:616-627, 1988; Rosenfeldet al., Science 252:431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci.USA 91:215-219, 1994; Kass-Eisler et al., Proc. Natl. Acad. Sci. USA90:11498-11502, 1993; Guzman et al., Circulation 88:2838-2848, 1993; andGuzman et al., Cir. Res. 73:1202-1207, 1993. Techniques forincorporating DNA into such expression systems are well known to thoseof ordinary skill in the art. The DNA may also be “naked,” as described,for example, in Ulmer et al., Science 259:1745-1749, 1993 and reviewedby Cohen, Science 259:1691-1692, 1993. The uptake of naked DNA may beincreased by coating the DNA onto biodegradable beads, which areefficiently transported into the cells.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will vary depending on the mode of administration.Compositions of the present invention may be formulated for anyappropriate manner of administration, including for example, topical,oral, nasal, intravenous, intracranial, intraperitoneal, subcutaneous,intradermal or intramuscular administration. For parenteraladministration, such as subcutaneous injection, the carrier preferablycomprises water, saline, alcohol, a fat, a wax or a buffer. For oraladministration, any of the above carriers or a solid carrier, such asmannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, sucrose, and magnesium carbonate, may beemployed. Biodegradable microspheres (e.g., polylactate polyglycolate)may also be employed as carriers for the pharmaceutical compositions ofthis invention. Suitable biodegradable microspheres are disclosed, forexample, in U.S. Pat. Nos. 4,897,268 and 5,075,109.

In addition, the carrier may contain other pharmacologically-acceptableexcipients for modifying or maintaining the pH, osmolarity, viscosity,clarity, color, sterility, stability, rate of dissolution, or odor ofthe formulation. Similarly, the carrier may contain still otherpharmacologically-acceptable excipients for modifying or maintaining thestability, rate of dissolution, release, or absorption or penetrationacross the blood-brain barrier of the OC related molecule. Suchexcipients are those substances usually and customarily employed toformulate dosages for parenteral administration in either unit dose ormulti-dose form or for direct infusion into the CSF by continuous orperiodic infusion from an implanted pump.

Such compositions may also comprise buffers (e.g., neutral bufferedsaline or phosphate buffered saline), carbohydrates (e.g., glucose,mannose, sucrose or dextrans), mannitol, proteins, polypeptides or aminoacids such as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.Alternatively, compositions of the present invention may be formulatedas a lyophilizate. Compounds may also be encapsulated within liposomesusing well known technology.

Any of a variety of adjuvants may be employed in the vaccines of thisinvention. Most adjuvants contain a substance designed to protect theantigen from rapid catabolism, such as aluminum hydroxide or mineraloil, and a stimulator of immune responses, such as lipid A, Bortadellapertussis or Mycobacterium tuberculosis derived proteins. Suitableadjuvants are commercially available as, for example, Freund'sIncomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit,Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.);aluminum salts such as aluminum hydroxide gel (alum) or aluminumphosphate; salts of calcium, iron or zinc; an insoluble suspension ofacylated tyrosine acylated sugars; cationically or anionicallyderivatized polysaccharides; polyphosphazenes biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such as GMCSF or interleukin-2, -7, or -12, may also be used as adjuvants.

Within the vaccines provided herein, the adjuvant composition ispreferably designed to induce an immune response predominantly of theTh1 type. High levels of Th1-type cytokines (e.g., IFN-α, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6, IL-10 and TNF-β) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of compound followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite, such as a site of surgical excision of a tumor. Sustained-releaseformulations may contain a polypeptide, polynucleotide or antibodydispersed in a carrier matrix and/or contained within a reservoirsurrounded by a rate controlling membrane. Carriers for use within suchformulations are biocompatible, and may also be biodegradable;preferably the formulation provides a relatively constant level ofactive component release. The amount of active compound contained withina sustained release formulation depends upon the site of implantation,the rate and expected duration of release and the nature of thecondition to be treated or prevented.

Antigen Presenting Cells

Any of a variety of delivery vehicles may be employed withinpharmaceutical compositions and vaccines to facilitate production of anantigen-specific immune response that targets tumor cells. Deliveryvehicles include antigen presenting cells (APCs), such as dendriticcells, macrophages, B cells, monocytes and other cells that may beengineered to be efficient APCs. Such cells may, but need not, begenetically modified to increase the capacity for presenting theantigen, to improve activation and/or maintenance of the T cellresponse, to have anti-tumor or anti-infective effects per se and/or tobe immunologically compatible with the receiver (i.e., matched BLAhaplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in titro) and based on the lack of differentiationmarkers of B cells (CD19 and CD20), T cells (CD3), monocytes (CD14) andnatural killer cells (CD56), as determined using standard assays.Dendritic cells may, of course, be engineered to express specific cellsurface receptors or ligands that are not commonly found on dendriticcells in vivo or ex vivo, and such modified dendritic cells arecontemplated by the present invention. As an alternative to dendriticcells, secreted vesicles antigen-loaded dendritic cells (calledexosomes) may be used within a vaccine (see Zitvogel et al., Nature Med.4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce maturation and proliferation of dendriticcells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor, mannose receptor and DEC-205marker. The mature phenotype is typically characterized by a lowerexpression of these markers, but a high expression of cell surfacemolecules responsible for T cell activation such as class I and class IINMC, adhesion molecules (e.g., CD54 and CD11) and costimulatorymolecules (e.g., CD40, CD80 and CD86).

APCs may generally be transfected with a polynucleotide encoding an OCpolypeptide (or portion or other variant thereof) such that the OCpolypeptide, or an immunogenic portion thereof, is expressed on the cellsurface. Such transfection may take place ex vivo, and a composition orvaccine comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and Cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the OC polypeptide, DNA (nakedor within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

Therapeutic and Prophylactic Methods

Treatment includes prophylaxis and therapy. Prophylaxis or therapy canbe accomplished by a single direct injection at a single time point ormultiple time points to a single or multiple sites. Administration canalso be nearly simultaneous to multiple sites. Patients or subjectsinclude mammals, such as human, bovine, equine, canine, feline, porcine,and ovine animals. The subject is preferably a human.

A cancer may be diagnosed using criteria generally accepted in the art,including the presence of a malignant tumor. Pharmaceutical compositionsand vaccines may be administered either prior to or following surgicalremoval of primary tumors and/or treatment such as administration ofradiotherapy or conventional chemotherapeutic drugs.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors or infected cells with theadministration of immune response-modifying agents (such as polypeptidesand polynucleotides disclosed herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells or antibodies) that candirectly or indirectly mediate antitumor effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8+cytotoxic T lymphocytes and CD4+ T-helper tumor-infiltratinglymphocytes), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. In a preferred embodiment, dendritic cells are modifiedin vitro to present the polypeptide, and these modified APCs areadministered to the subject. T cell receptors and antibody receptorsspecific for the polypeptides recited herein may be cloned, expressedand transferred into other vectors or effector cells for adoptiveimmunotherapy. The polypeptides provided herein may also be used togenerate antibodies or anti-idiotypic antibodies (as described above andin U.S. Pat. No. 4,918,164) for passive immunotherapy.

Administration and Dosage

The compositions are administered in any suitable manner, often withpharmaceutically acceptable carriers. Suitable methods of administeringcells in the context of the present invention to a subject areavailable, and, although more than one route can be used to administer aparticular cell composition, a particular route can often provide a moreimmediate and more effective reaction than another route.

The dose administered to a patient, in the context of the presentinvention, should be sufficient to effect a beneficial therapeuticresponse in the patient over time, or to inhibit disease progression.Thus, the composition is administered to a subject in an amountsufficient to elicit an effective immune response to the specificantigens and/or to alleviate, reduce, cure or at least partially arrestsymptoms and/or complications from the disease. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”

Routes and frequency of administration of the therapeutic compositionsdisclosed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions and vaccines may beadministered, by injection (e.g., intracutaneous, intratumoral,intramuscular, intravenous or subcutaneous), intranasally (e.g., byaspiration) or orally. Preferably, between 1 and 10 doses may beadministered over a 52 week period. Preferably, 6 doses areadministered, at intervals of 1 month, and booster vaccinations may begiven periodically thereafter. Alternate protocols may be appropriatefor individual patients. In one embodiment, 2 intradermal injections ofthe composition are administered 10 days apart.

A suitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored, for example, by measuring the anti-tumorantibodies in a patient or by vaccine-dependent generation of cytolyticeffector cells capable of killing the patient's tumor cells in vitro.Such vaccines should also be capable of causing an immune response thatleads to an improved clinical outcome (e.g., more frequent remissions,complete or partial or longer disease-free survival) in vaccinatedpatients as compared to nonvaccinated patients. In general, forpharmaceutical compositions and vaccines comprising one or morepolypeptides, the amount of each polypeptide present in a dose rangesfrom about 100 μg to 5 mg per kg of host. Suitable volumes will varywith the size of the patient, but will typically range from about 0.1 mLto about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a tumor protein generally correlate with an improvedclinical outcome. Such immune responses may generally be evaluated usingstandard proliferation, cytotoxicity or cytokine assays, which may beperformed using samples obtained from a patient before and aftertreatment.

EXAMPLES

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Example 1 Identification of Biomarkers for Ovarian Cancer Using StrongAnion-Exchange Protein Chips

This example demonstrates three ovarian cancer biomarker protein panelsthat, when used together, effectively distinguished serum samples fromhealthy controls and patients with either benign or malignant ovarianneoplasia. In summary, 184 serum samples from patients with ovariancancer (n=109), patients with benign tumors (n=19), and healthy donors(n=56) were analyzed on strong anion-exchange surfaces usingsurface-enhanced laser desorption/ionization time-of-flight massspectrometry technology. Univariate and multivariate statisticalanalyses applied to protein-profiling data obtained from 140 trainingserum samples identified three biomarker protein panels. The first panelof five candidate protein biomarkers, termed the screening biomarkerpanel, effectively diagnosed benign and malignant ovarian neoplasia(95.7% sensitivity, 82.6% specificity, 89.2% accuracy, and receiveroperating characteristic (ROC) area under the curve of 0.94). The othertwo panels, consisting of five and four candidate protein biomarkerseach, effectively distinguished between benign and malignant ovarianneoplasia and were therefore referred to as validation biomarker panel I(81.5% sensitivity, 94.9% specificity, 88.2% accuracy, and ROC=0.94) andvalidation biomarker panel II (72.8% sensitivity, 94.9% specificity,83.9% accuracy, and ROC=0.90). The three ovarian cancer biomarkerprotein panels correctly diagnosed 41 of the 44 blinded test samples: 21of 22 malignant ovarian neoplasias (10 of 11 early-stage ovarian cancer(I/II) and 11 of 11 advanced-stage ovarian cancer (III/IV)), 6 of 6 lowmalignant potential, 5 of the 6 benign tumors, and 9 of 10 normalpatient samples.

The following abbreviations are used in this example: CA125, ovariancancer antigen 125; LMP, low malignant potential; ROC, receiveroperating characteristic; SBP, screening biomarker panel; SAX2, stronganion-exchange; SELDI-TOF-MS, surface-enhanced laserdesorption/ionization time-of-flight MS; VBP, validation biomarkerpanel.

Methods

Materials. Serum samples were obtained from healthy individuals (n=56),patients with ovarian cancer (n=109), and patients with benign tumors(n=19) through the Gynecological Oncology Group and Cooperative HumanTissue Network. Serum samples had been collected preoperatively frompatients with malignant and benign ovarian tumors. Sample numbers usedfor profiling (training group) and validation (test group) are listedaccording to histopathology in Table 1, and the stage and grade oftumors from patients with ovarian cancer are listed in Table 2. TABLE 1Sample number used in training and test groups according tohistopathology Histopathology Training group Test group Total Normal 4610 56 Benign 13 (7S, 6M) 6 (1S, 4M, 1A) 19 Adenocarcinoma 14 (7S, 7M) 6(2S, 3M, 1A) 20 of LMP Adenocarcinoma 67 (43S, 7M, 22 (12S, 2M, 2A, 897A, 8E, 2C) 5E, 1C) Total 140 44 184S, serous; M, mucinous; A, S or M; E, endometrioid; C, clear cell.

TABLE 2 Stage vs. grade of adenocarcinoma samples Adenocarcinoma of theovary Stage vs. Grade Grade Grade Grade Un- grade LMP 1 2 3 4 knownTotal Stage I 18 10 12 2 2 0 44 Stage II 1 0 7 4 0 0 12 Stage III 1 7 1211 14 7 52 Stage IV 0 0 0 0 1 0 1 Total 20 17 31 17 17 7 109

Preparation of Serum Samples for SELDI Analysis. Two different dilutions(1:4 and 1:25) of serum samples were processed on strong anion-exchange(SAX2) chips according to the manufacturer's protocols (CiphergenBiosystems). Briefly, the array spots were preactivated with bindingbuffer (1× PBS/0.1% Triton X-100, pH 7.5) at room temperature for 15 minin a humidifying chamber. Each serum sample was first diluted 1:2 or 1:5with 9 M urea/2% Chaps/50 mM Tris HCl, pH 9.0, and was further diluted1:2 or 1:5, respectively, in binding buffer. Three microliters of eachdiluted sample was spotted onto preactivated SAX2 protein array chipsand incubated in a humidity chamber for 30 min at room temperature. Thechips were washed twice with binding buffer and once with HPLC H₂O, andthen air-dried. The chips were then sequentially treated with sinapinicacid (3,5-ditnethoxy-4-hydroxycinnamic acid), first with 0.6 μl of a100% saturated solution followed by 0.8 μl of a 50% saturated solution.The sinapinic acid solution was 50% acetonitrile and 0.5%trifluoroacetic acid. The chips were analyzed with the CiphergenProteinChip Reader (model PBSII). Each dilution was analyzed separatelyto confirm reproducibility in identifying the differentially expressedproteins.

Ciphergen ProteinChip SELDI-TOF-MS Analysis. The arrays were analyzedwith the Ciphergen ProteinChip Reader (model PBSII). The mass spectra ofproteins were generated by using an average of 65 laser shots at a laserintensity of 230-280 arbitrary units. For data acquisition of lowmolecular weight proteins, the detection size range was between 2 and 18kDa, with a maximum size of 25 kDa. The laser was focused at 10 kDa. Thedetector sensitivity was set at 8, and the laser intensity was set at230 for the 1:4 and 250 for the 1:25 dilution. For the high molecularweight proteins, the detection size range was between 20 and 150 kDa,with a maximum size of 250 kDa. The laser was focused at 85 kDa. Thedetector sensitivity was set at 9, and the laser intensity was set at260 for the 1:4 dilution and 280 for the 1:25 dilution. Themass-to-charge ratio (m/z) of each of the proteins captured on the arraysurface was determined according to externally calibrated standards(Ciphergen Biosystems, Fremont, Calif.): bovine insulin (5,733.6 Da),human ubiquitin (8,564.8 Da), bovine cytochrome c (12,230.9 Da), bovinesuperoxide dismutase (15,591.4 Da), bovine β-lactoglobulin A (18,363.3Da), horseradish peroxidase (43,240 Da), BSA (66,410 Da), and chickenconalbumin (77,490 Da).

Statistical Analysis. The data were analyzed with PROTEINCHIP dataanalysis software version 3.0 (Ciphergen Biosystems). For eachcomparison, the raw intensity data were normalized by using the totalion current of all profiles in the groups compared. The peak intensitieswere normalized to the total ion current of m/z between 3,000 and 25,000Da for the low molecular weight range and between 4,000 and 250,000 Dafor the high molecular weight range. The test group (n=44) wasnormalized to all 140 training samples before using their intensitiesagainst the statistically derived intensity cutoffs. The BiomarkerWizard application (nonparametric calculations; Ciphergen Biosystems)was used to compile all spectra and autodetect quantified mass peaks.Peak labeling was completed by using second-pass peak selection with0.3% of the mass window, and estimated peaks were added. Samplestatistics were performed on groups of profiles (normal vs.benign/cancer and normal/benign vs. cancer). Protein differences (foldchanges) were calculated among the various groups. A protein wasconsidered differentially expressed in the ovarian cancer groups if,when compared with the normal group, statistically significantdifferences in its intensity were observed (p≦0.01) in both the 1:4 andthe 1:25 dilution analysis. Using the intensities derived from the 1:25dilution analysis, univariate comparisons of marker intensity summarystatistics of representative markers were performed using statisticalanalysis software (SAS, Version 8.0, SAS Institute, Cary, N.C.). Foreach marker, t tests and Wilcoxon rank sum tests were used to comparethe mean and median standardized intensities, respectively, between thenormal and cancer groups and to determine their corresponding P values(nonparametric for medians). The SAS program was used to determine the“best” intensity cutoff for each marker at either highest accuracy orwhen sensitivity equals specificity. Receiver operating characteristic(ROC) curves (plot of sensitivity vs. (1-specificity) for each possiblecutoff) were generated for proteins with low P values, and the highestindividual diagnostic power was calculated by using SAS. Multivariatelogistic regression analysis was performed on the biomarkers using SAS.The program was used to analyze various combinations of markers givingpredictive scores for each panel tested. This predictive score is a sumof the individually weighted marker intensities.

Results

Identification of Differentially Expressed Ovarian Cancer-AssociatedProteins in Serum. Using SELDI-TOF-MS proteomics technology (CiphergenBiosystems), proteins differentially expressed between serum fromhealthy individuals (n=46) and serum from patients with ovarian cancer(benign, n=13; low malignant potential (LMP), n=14; and malignant, n=67)were identified. A representative pseudogel view of specific candidateovarian cancer tumor markers and a spectral overlay of candidate markersfrom healthy vs. diseased individuals are shown in FIG. 1A and 1B.Statistical analysis on potential biomarkers with the lowest P values(≦0.01). The P values were generated with nonparametric tests from boththe BIOMARKER WIZARD application (Ciphergen Biosystems) and the SASprogram. Sensitivity, specificity, overall accuracy, and ROC area valueswere computed for each biomarker using the SAS program.

Neoplasia Biomarkers and Malignant Neoplasia Biomarkers. Proteins thatare differentially expressed in healthy individuals vs. patients withany ovarian tumor, including benign or malignant, were considered first.Under the conditions tested, 10 biomarkers were identified (4.1 kDa, 4.4kDa, 7.7 kDa, 12.9 kDa, 15.1 kDa, 15.9 kDa, 18.9 kDa, 23.0 kDa, 30.1kDa, and 53.5 kDa) and classified as “neoplasia biomarkers” (markersbest at identifying benign and malignant samples). All markers, exceptmarker 12.9 kDa, showed increased expression in patients with ovariancancer. At highest accuracy, the individual markers in the neoplasiabiomarkers group had ROC area values ranging from 0.711 to 0.833,sensitivities from 60.6% to 84.0%, specificities from 52.2% to 89.1%,and accuracies from 67.1% to 78.5%. Proteins that are differentiallyexpressed in healthy individuals and individuals with benign tumors vs.patients with malignant ovarian tumors were examined next. Under theconditions tested, 13 proteins (3.1 kDa, 4.5 kDa, 5.1 kDa, 7.8 kDa, 8.2kDa, 13.9 kDa, 16.9 kDa, 18.6 kDa, 21.0 kDa, 28.0 kDa, 79.0 kDa, 93.0kDa, and 106.7 kDa) were identified and grouped as “malignant neoplasiabiomarkers” (markers best at identifying malignant samples). Markers 3.1kDa, 4.5 kDa, 5.1 kDa, 7.8 kDa, 8.2 kDa, 16.9 kDa, and 18.6 kDa showedincreased expression in patients with ovarian cancer, whereas markers13.9 kDa, 21.0 kDa, 28.0 kDa, 79.0 kDa, 93.0 kDa, and 106.7 kDa showeddecreased expression in patients with ovarian cancer. The individualproteins in the malignant biomarkers group had values for ROC arearanging from 0.617 to 0.851, sensitivities from 48.1% to 81.5%,specificities from 66.1% to 88.1%, and accuracies from 61.3% to 79.3%.

Biomarker Panels. Multivariate analysis was performed on both theneoplasia and malignant neoplasia groups, separately, to identify panelsof biomarkers that will diagnose ovarian neoplasm (benign or malignant)or distinguish between benign and malignant ovarian tumors. The SASprogram, through a process of statistical logistic backward eliminationto avoid “overfitting” bias, produced panels with the least redundantmarkers. Thus, these markers were excluded from the panel. Multivariateanalysis of the 10 candidate ovarian neoplasia biomarkers resulted in anovarian cancer screening biomarker panel (SBP) of five markers (4.4 kDa,15.9 kDa, 18.9 kDa, 23.0 kDa, and 30.1 kDa) with a collective ROC area(0.94; FIG. 2A) higher than the best individual ovarian neoplasiadiagnostic biomarker (0.83; 15.1 kDa). The sensitivity, specificity, andoverall accuracy for the SBP were 95.7%, 82.6%, and 89.2%, respectively(Table 3 and FIG. 2B). Similarly, multivariate analyses of the 13malignant neoplasia biomarkers yielded two independent panels:validation biomarker panel (VBP) I of five markers (3.1 kDa, 13.9 kDa,21.0 kDa, 79.0 kDa, and 106.7 kDa) and VBP II of four markers (5.1 kDa,16.9 kDa, 28.0 kDa, and 93.0 kDa) with ROC area values (0.94 and 0.90;Table 3 and FIG. 3A and 3C) higher than the best individual malignantovarian neoplasia diagnostic biomarker (0.85; 79.0 kDa). Thesensitivity, specificity, and overall accuracy values for the VBPs were81.5%, 94.9%, and 88.2% for panel I and 72.8%, 94.9%, and 83.9% forpanel II, when maximizing overall accuracy (Table 3 and FIG. 3B and 3D).TABLE 3 Statistical summary of biomarker panels Sensi- Speci- Accu-Ovarian cancer tivity, % ficity, % racy,* % ROC panels A B A B A B AreaScreening 86.2 95.7 87.0 82.6 86.6 89.2 0.94 biomarker panel Validation85.2 81.5 84.7 94.9 85.0 88.2 0.94 biomarker panel I Validation 81.572.8 81.4 94.9 81.4 83.9 0.90 biomarker panel IIA values are represented when sensitivity equals specificity. B valuesare represented at their highest accuracy.*Accuracy is 0.5 sensitivity + 0.5 specificity

Screening and Validation of Test Samples. The SBP and the VBPs weretested on 44 blinded test samples (10 normal, 6 benign, 6 LMP, and 22invasive ovarian cancers, and 11 early-stage (I/II) and 11advanced-stage (III/IV) carcinomas). For the ovarian cancer SBP, thetest sensitivity (number benign and cancer correctly labeledpositive/number true positive) at highest accuracy was 91.2%, the testspecificity (number normal correctly labeled negative/number truenegative) was 80%, and test accuracy was 85.6%. For early-stage (I/II),the SBP using the highest accuracy cutoff resulted in a sensitivity of100% (identified 11 of 11), advanced-stage (III/IV) sensitivity of 100%(identified 11 of 11), benign tumor sensitivity of 50% (3 of 6), and anontumor specificity of 80% (identified 8 of 10 nondiseased). For theovarian cancer VBP I, the overall test sensitivity, specificity, andaccuracy were 71.4%, 100%, and 85.7%, respectively, and resulted in testsensitivity for earlystage (I/II) of 54.5% (identified 6 of 11), and foradvanced-stage (III/IV) of 72.7% (identified 8 of 11), benign tumorspecificity of 100% (identified 6 of 6), and nontumor specificity of100% (identified 10 of 10). Finally, for the ovarian cancer VBP II, theoverall test sensitivity, specificity, and accuracy were 89.3%, 56.3%,and 72.8%, respectively, and resulted in test sensitivity forearly-stage (I/II) of 90.9% (identified 10 of 11) and 81.8% foradvanced-stage (III/IV; identified 9 of 11), with benign tumorspecificity of 50% (identified 3 of 6) and nontumor specificity of 60%(identified 6 of 10). Predictions made with all three panels together,by using their score thresholds at highest accuracy, correctly diagnosed41 of 44 test samples: 21 of 22 malignant carcinomas (10 of 11early-stage (I/II), 11 of 11 advanced-stage (III/IV)), 6 of 6 LMP, 5 ofthe 6 benign tumors, and 9 of 10 normal patient samples.

Example 2 Characterization of Ovarian Cancer Serum Biomarkers For EarlyDetection

This example demonstrates the identification, characterization, andvalidation of the proteins that represent the SELDI-TOF-MS peaks fromthe ovarian cancer biomarker panels. Mass spectrometry and otheranalytical methods were employed to identify proteins of interest inhuman serum. The Example describes the identity of proteins thatrepresent the m/z peaks 12.9, 13.8, 15.1, 15.9, 28 and 78.9 kDa in theovarian cancer biomarker panels. Using micro-liquidchromatography-tandem mass spectrometry, the following m/z peaks wereidentified as: transthyretin (TTR): 12.9 kDa and 13.9 kDa, hemoglobin,both alpha-hemoglobin (alpha-Hb): 15.1 kDa, and beta-hemoglobin(beta-Hb): 15.9 kDa, apolipoprotein AI (ApoAI): 28 kDa and transferrin(TF): 78.9 kDa. Western and ELISA techniques (independent of SELDI)confirmed the differential expression of TTR, Hb and TF in a group ofovarian cancer serum samples. Multivariate analyses improved thedetection of early stage ovarian tumors flow mallignant potential andmalignant) as compared to cancer antigen CA125 alone. Multivariateanalysis with only the mucinous subtype of early stage ovarian tumorsshowed the marker to greatly improve the detection of disease ascompared to CA125 alone.

This Example uses the following abbreviations: CA125, ovarian cancerantigen 125; Hb, hemoglobin; LMP, low malignant potential; μLC-MSMS,micro-liquid chromatography-tandem mass spectrometry; ROC, receiveroperating characteristic; SAS, statistical analysis software; SAX2,strong anion exchange; SELDI-TOF-MS, Surface-Enhanced LaserDesorption/Ionization Time-of-Flight Mass Spectrometry; TF, transferrin;TTR, tranthyretin.

Materials

Serum samples were obtained through the Gynecological Oncology Group andCooperative Human Tissue Network and had been collected preoperatively.Purified protein preparations of transthyretin (TTR), hemoglobin (Hb),transferrin (TF) and apolipoprotein AI (ApoAI), were purchased fromSigma-Aldrich (St. Louis, Mo.).

Serum Protein Fractionation

Serum (30 ul) was desalted on P-6 Micro Bio-Spin chromatography columns(Bio-Rad, Hercules Calif.) and dealbuminized using Affi-Gel Blue Gel(Bio-Rad, Hercules, Calif.) in macro columns according to themanufacturer's protocol. Serum was fractionated using anion exchangespin columns (Ciphergen, Fremont, Calif.), by eluting with a series ofbuffers decreasing in pH (20 mM sodium phosphate, pH 7.0 and 6.3; 50 mMsodium acetate, pH 5.0 and 4.0; 100 mM sodium citrate, pH 2.5 and 2.3with or without 1M sodium chloride). Protein fractions were analyzed onstrong anion exchange (SAX2) chips using a SELDI-TOF-MS PSII as reportedpreviously.

Serum Protein Purification, Passive Elution and Confimation bySELDI-TOF-MS

Fractions confirmed by SELDI-TOF-MS to contain a significant majority ofthe 13.9 kDa peak using either an NP20 or Au chip according to themanufacturer's protocol (Ciphergen, Fremont Calif.) were pooled anddried by centrifugal evaporation. Fractions containing the 15.9 and 79kDa proteins were further purified on SDS-PAGE. The gels were thenstained with Simply Blue Safestain (Invitrogen, Carlsbad, Calif.), bandswith molecular weights corresponding to the markers were excised, gelslices were cut in half, and half of the gel slice was passively elutedaccording to Le Bihan et al. (17). Briefly, the gel was dehydrated withacetonitrile for ≧10 min, dried in a heat block for 10 min at 42° C. andrehydrated in 50% formic acid/25%acetonitrile/15% isopropyl alcohol.Tubes were placed in a sonicator bath for 30 min followed by vortexingfor 1 h. A fraction of the eluate was applied onto an NP20 or Au chipand profiles were compared to regions of serum profiles corresponding tomarkers 15.9 and 79 kDa. The 28 kDa protein was purified fromdealbuminized serum using PHM-L Liposorb (Calbiochem, San Diego Calif.),followed by SDS-PAGE. Protein from half of the gel slide was passivelyeluted and confirmed by SELDI-TOF-MS.

Tryptic Digestion and μLC-MSMS

Concentrated pooled anion exchange fractions containing the 13.9 kDamarker were reduced (10 mM DTT in 50 mM ammonium bicarbonate; 30 min,24° C.), alkylated (55 mM iodoacetamide in 50 mM ammonium bicarbonate;20 min, 24° C.), and treated with trypsin (Promega; 6 ng/μl in 50 mMammonium bicarbonate; 3 h, 37° C.). After confirming that the passivelyeluted proteins from half of the SDS-PAGE gel slice corresponded to the15.9, 28 and 79 kDa markers, the remaining half was used for in geltryptic digest. Briefly, the gel slices were destained in 200 mMammonium bicarbonate/40% acetonitrile, washed with a 1:1 mixture of 100mM ammonium bicarbonate: acetonitrile for 10 min and dehydrated withacetonitrile 10 min. After vacuum drying 5 min, the gel slices werereduced with 10 mM DTT in 50 mM ammonium bicarbonate for 60 min at 60°C. After cooling, gel slices were incubated for 45 min at 45° C. with 50mM iodoacetic acid in 50 mM ammonium bicarbonate. After washing anddehydrating the gel slices with 100 mM ammonium bicarbonate andacetonitrile for 10 min, they were vacuum dried and tryptic digestionperformed with 50 mM ammonium bicarbonate containing 10 ng/ml trypsin inan ice bath for 45 min. Additional 50 mM ammonium bicarbonate was addedand digestion was continued overnight at 37° C. Peptides were recoveredby saturating the gel slices with HPLC grade water and extracted with50% acetonitrile containing 1% trifloroacetic acid three times for 10min each. Extracts were dried in a cold speedvac for 1 hr. Dried sampleswere analyzed by micro-liquid chromatography-tandem mass spectrometry(μLC-MSMS) as described previously (18) and the data was used to searchhuman databases using Sonar ms/ms™ (Genomic Solutions, Ann Arbor Mich.)and TurboSEQUEST™ (Thermo Electron Corp., San Jose Calif.). Briefly,samples were analyzed by μLC-MSMS with data-dependent acquisition(LCQ-DECA, ThermoFinnigan, San Jose, Calif.) after dissolution in 5 μlof 70% acetic acid (v/v). A reverse-phase column (200 μm×10 cm, PLRP/S 5μm, 300 Å; Michrom Biosciences, San Jose, Calif.) was equilibrated with95% A, 5% B (A, 0.1% formic acid in water; B, 0.10% formic acid inacetonitrile) and a linear gradient was initiated ramping to 60% A, 40%B after 50 min and 20% 0.1% A, 80% B after 65 min. Column eluent wasdirected to a coated glass electrospray emitter (TaperTip,TT150-5050-CE-5, New Objective) at 3.3 kV for ionization withoutnebulizer gas. The mass spectrometer was operated in “triple-play” modewith a survey scan (400-1500 m/z), data-dependent zoom scan, and MSMS.

Western Blot Analysis

Total serum protein was determined by Bradford assay using BSA as thestandard (Sigma-Aldrich, St. Louis Mo.) and equal protein amounts(0.1-30 μg) or volume (1 μl) was loaded onto SDS-PAGE gels. Westernanalyses were performed as described previously (19). Rabbit anti-TTRantibody was purchased from DAKO (Carpinteria, Calif.).

Goat anti-Hb was obtained from Bethyl Laboratories Inc. (Montgomery,Tex.), goat anti-ApoAI from Biodesign international (Saco, Me.), andgoat anti-seroTF was from Abcam Inc. (Cambridge, Mass.). Secondaryantibodies, anti-goat IgG and anti-rabbit IgG (both conjugated to HRP),were used at a 1:4000 dilution.

Immunoprecipitation

Serum (-250 μg) was pre-cleared in immunoprecipitation buffer (1% TritonX 100/0.025% sodium azide/0.1M NaCl/0.05M Tris-HCl, pH7.5/5 mM EDTA andprotease inhibitors) with 6% v/v A/G Plus agarose beads (Sigma-Aldrich,St. Louis Mo.) for 3 h at 4° C. with tumbling. Beads were removed bycentrifugation for 30 s at 13.2K rpm, antibodies were added to thesupernatant and rotated at 4° C. overnight. The antibody/antigencomplexes were precipitated by adding 6% v/v A/G Plus agarose beads for3.5 h, at 4° C. Beads were removed from depleted serum by centrifugationfor 30 s at 13.2K rpm. Post pre-cleared and depleted samples wereanalyzed on SELDI-TOF-MS with SAX2 chips as previously reported.

ELISA

For TTR and ApoAI, optimized dilutions of pure proteins and serumsamples were coated onto 96-well Immobilon plates overnight at 4° C. incoating buffer (0.1M carbonate buffer, pH 9.6; 1:10,000 for TTR;1:10,000 for ApoAI). After washing, plates were blocked with 0.05% Tween20/0.25% BSA/1× PBS at RT for 1 h. Primary TTR antibody was used at a1:10,000 dilution and secondary antibody (rabbit-HRP) was used at a1:5,000 dilution. Primary ApoAI antibody was used at a 1:10,000 dilutionand secondary antibody (goat-HRP) was used at a 1:5,000 dilution.Detection was performed with TMB (KPL, Gaithersburg, Md.) and stoppedwith 0.5M sulfuric acid. Hb and TF ELISA kits were purchased from BethylLaboratories Inc. (Montgomery, Tex.) and used according to themanufacturer's protocols with optimized serum and antibodyconcentrations. Hb capture antibody was used at a 1:100 dilution, HRPdetection antibody at a 1:10,000 dilution, while serum was diluted1:10,000. TF capture antibody was used at a 1:100 dilution, HRPdetection antibody at a 1:150,000 dilution and the serum was diluted1:50,000. The CA125 ELISA kit was purchased from BioCheck (Burlingame,Calif.) and used according to the manufacturer's protocol. Plates wereread in a Kinetic microplate reader (Molecular Devices, SunnyvaleCalif.) at 450 nm and analysis was performed using the SoftMax Pro v4.3LS software (Molecular Devices, Sunnyvale Calif.).

Statistical Analysis

SELDI-TOF-MS data was analyzed with Ciphergen's ProteinChip dataanalysis software version 3.0 (Ciphergen Biosystems, Fremont Calif.).SELDI-TOF-MS and ELISA marker intensities were analyzed usingstatistical analysis software (SAS, Version 8.0, SAS Institute, CaryN.C.). The Mann-Whitney non-parametric two-tailed t-test with 95%confidence interval was used to determine p-values.

Results

Identification of potential candidate proteins using size, pI andTagIdent. From the pool of markers reported previously (16), we chosefive biomarker proteins (12.9, 13.9, 15.9, 28.0, and 79 kDa) for thecurrent studies; 12.9, 13.9, 28.0, and 79 decreased in patients withovarian cancer whereas 15.9 increased in patients with ovarian cancer.We performed an online TagIdent (protein database) search using the sizedetermined from SELDI-TOF-MS analysis and the corresponding pI asdetermined by anion exchange fractionation (FIG. 4). The m/z values usedfor the searches were 12785, 13797, 15850, 27977, and 78715,corresponding to markers, 12.9, 13.9, 15.9, 28 and 79 respectively.Using search criteria allowing for a 0.5% size error and ±2 pI range, weidentified TTR, beta-Hb, and ApoAI, as potential candidate proteins form/z peaks 13.9, 15.9, and 28 respectively. The m/z peak 79 and itscorresponding pI did not result in any candidate proteins in ouranalysis, however; recently a similar 79 kDa SELDI peak from a separateovarian cancer profiling study was identified as TF.

The SELDI-TOF-MS profiles of purified TTR, Hb, ApoAI and sero-TF matchedwith the 12.9, 13.9, 15.1, 15.9, 28 and 79 m/z peaks from the humanserum. We purchased purified (from human serum) preparations of thecandidate proteins and compared their SELDI-TOF-MS profiles to thosegenerated from serum samples for 12.9, 13.9, 15.9, 28 and 79 (FIG. 5).Pure TTR protein resulted in peaks with an m/z similar to that obtainedfor both 12.9 and 13.9 kDa serum markers. Pure Hb gave a peak of m/zsimilar to the 15.9 kDa serum marker, likely corresponding to the betachain, while a second peak from the pure Hb matched with one of ouroriginal (16) non-panel serum markers (15.1 kDa) likely correlating withthe alpha subunit of Hb. Pure ApoAI protein resulted in a peak with anm/z similar to that obtained for the 28 kDa serum marker, while the peakfrom pure sero-TF aligned with the 79 kDa marker peak.

Fractionation and tryptic peptide fragmentation and analysis by tandemmass spectrometry confirmed the identities of biomarker proteins as TTR,Hb, ApoAI and TF. To further confirm the identities of the fivebiomarkers, the peaks corresponding to the respective sizes werepartially purified from serum following dealbuminization and anionexchange chromatography (FIG. 4). The partially purified proteins weresubjected to tryptic digestion followed by μLC-MSMS analysis and theresulting fragments were searched against human protein databases (Sonarand SEQUEST). The results confirmed the 13.9 kDa protein as TTR, beta-Hbas the 15.9 kDa protein, ApoAI as the 28 kDa protein and TF as the 79kDa protein. We also confirmed the non-panel 15.1 kDa marker asalpha-Hb. Due to limitations in obtaining sufficient quantities we didnot perform μLC-MSMS analysis on the 12.9 kDa marker. However, a similar12.9 kDa SELDI-TOF-MS peak has been recently reported to be a fragmentof TTR by purification and mass spectrometry analysis. Moreover, we havealso observed that pure TTR protein contains two peaks with m/z ratiossimilar to that obtained for 12.9 and 13.9 kDa serum markers (FIG. 5)suggesting that the 12.9 kDa peak is indeed a TTR fragment.

Immunodepletion studies further validated TTR, alpha-Hb, beta-Hb and TFas the proteins corresponding to the 13.9, 15.1, 15.9 and 79 kDa peaks.We next performed immunoprecipitation studies on pre-cleared normalserum to ensure that the SELDI-TOFMS peaks of the five markerscorrespond to the proteins identified by the methods described above.Immunoprecipitation of TTR and TF from pre-cleared normal serum resultedin depletion of the 13.9 kDa and 79 kDa protein (FIG. 6).Immunoprecipitation of Hb from pre-cleared serum derived from an ovariancancer patient, resulted in depletion of the 15.1 and 15.9 kDa proteins(FIG. 6).

Westerns confirmed TTR, Hb, and TF to be differentially expressed inserum from ovarian cancer patients. Using specific antibodies, wefurther confirmed the differential expression of TTR, Hb, and TF inserum samples from normal individuals, and patients with early stage(I/II) or late stage (III/IV) ovarian cancer by Western blotting.Membranes probed with anti-TTR or anti-sero-TF showed a decrease inprotein levels (FIG. 7), while anti-Hb antibody showed an increase inexpression between normal, early and late stage ovarian cancer (FIG. 7).No significant differential expression was observed for ApoAI on Westernblots.

ELISA experiments validated TTR, Hb, ApoAI and TF as biomarkers forovarian tumors. Differential expression of TTR, Hb, ApoAI and TF wasfurther validated in ELISA experiments performed with 27 normal samples,11 ovarian low malignant potential tumor (LMP) samples and 19 earlystage malignant ovarian tumor samples (FIG. 8A-8E). For the early stagemalignant ovarian tumors, the TTR concentration was approximately 175±25g/ml in the normal serum, while it was only 91±12 in the early stageovarian cancers (FIG. 8A). Interestingly, Swiss-protein databaseindicates normal levels of TTR to be between 100-400 μg/ml. Hb (FIG. 8B)indicated a significant difference between normal serum (0.11±0.01mg/ml) and early stage serum (0.35±0.12). The ApoAI concentration wasapproximately 1.21±0.41 mg/ml in the normal serum, while it was only0.33±0.02 mg/ml in the early stage ovarian cancers (FIG. 8C).Swiss-protein database indicates normal levels of ApoAI to be 0.9-2.1mg/ml. In the malignant samples tested, the concentration for TF (FIG.8D) was 2.82±0.12 mg/ml for normal and 1.88±0.19 mg/ml for early stage.Swiss-protein database indicates normal levels of TF to be 2-4 mg/ml.All antibodies, except for TTR, had been tested to work in ELISA assays.

Statistical analysis of ELISA data from normal and early stage ovariantumors identify TTR, Hb, ApoAI and TF as markers for early detection ofovarian tumors. The differential expression of our proteins betweennormal samples and LMP tumors was determined to be statisticallysignificant for all markers (FIG. 8A-8E). The differential expressionbetween normal samples and early stage tumor samples was significant forall individual markers, except Hb (FIG. 8B). We performed multivariateregression analysis with the ELISA data generated from the LMP and themalignant tumors and compared sensitivity, specificity and ROC valuesfrom our characterized markers, together, to CA125 alone or incombination with our markers (Table 4a, b). Table 4a and b indicatesensitivity and specificity, when they are given equal importance, andROC values of multivariate regression analysis of our markers for LMPtumors, early stage malignant tumors and LMP and malignant tumors asgenerated by SAS. Results show that our markers, combined with CA125,improve sensitivity, specificity and ROC values for all histologicalgroups (serous papillary, mucinous, endometrioid, and clear cell; Table4a) as well as for the mucinous subgroup (Table 4b). TABLE 4aMultivariate analysis of marker ELISA values for detection of earlystage ovarian tumors in all histological groups. Normal Sensitivity &ROC n = 27 Markers Specificity* (%) Area LMP CA125 ˜64% 0.758 (n = 11)TTR, Hb, ApoAI, TF ˜82% 0.953 TTR, Hb, ApoAI, TF, CA125 ˜82% 0.949Malignant CA125 ˜85% 0.875 (n = 19) TTR, Hb, ApoAI, TF ˜85% 0.920 TTR,Hb, ApoAI, TF, CA125 ˜89% 0.971 LMP & CA125 ˜78% 0.833 Malignant TTR,Hb, ApoAI, TF ˜86% 0.933 (n = 30) TTR, Hb, ApoAI, TF, CA125 ˜86% 0.959*Values are represented when threshold cutoffs are set where sensitivityand specificity are given equal importance

TABLE 4b Multivariate analysis of marker ELISA values for detection ofearly stage ovarian tumors of the mucinous histological subgroup NormalSensitivity & ROC n = 27 Markers Specificity* (%) Area LMP CA125 ˜51%0.562 (n = 6) TTR, Hb, ApoAI, TF ˜81% 0.926 TTR, Hb, ApoAI, TF, CA125˜84% 0.932 Malignant CA125 ˜67% 0.728 (n = 3) TTR, Hb, ApoAI, TF ˜100% 1.000 TTR, Hb, ApoAI, TF, CA125 ˜100%  1.000 LMP & CA125 ˜56% 0.613Malignant TTR, Hb, ApoAI, TF ˜87% 0.959 (n = 9) TTR, Hb, ApoAI, TF,CA125 ˜87% 0.955*Values are represented when threshold cutoffs are set where sensitivityand specificity are given equal importanceDiscussion

We reported the identification of several ovarian cancer biomarkersgenera~ted using Ciphergen's ProteinChip technology. When used aspanels, these markers resulted in improved sensitivity and specificityfor the detection of early stage ovarian cancer. In this example, wereport the identification of proteins that represent the previouslyreported biomarkers with m/z ratios 12.9, 13.9, 15.9, 28 and 79 kDa as aTTR fragment, TTR, beta-Hb, ApoAI and TF, respectively. We alsoidentified a non-panel marker, 15.1 kDa, as alpha-Hb. We have shown thattogether, TTR, Hb, ApoAI, TF and CA125 can improve ovarian tumor (allhistological subgroups) detection sensitivity by 8% when compared toCA125 alone (threshold cutoff where sensitivity and specificity areequally important). More interestingly, TTR, Hb, ApoAI, TF, and CA125,together improved sensitivity of detecting mucinous tumors by 31% overCA125 alone.

The detection sensitivities for our markers when tested against a poolof serum samples that contained all histological groups of ovariancancer are similar to those reported by other investigators. Rai et al.identified and purified transferrin, immunoglobulin heavy chain and afragment of the haptoglobin precursor protein as candidate biomarkers ofovarian cancer using SELDI-TOF-MS technology with nickel-coatedimmobilized metal affinity capture type 3 arrays (IMAC3). Statisticalanalysis of these data demonstrated that the diagnostic index combiningtwo of the biomarkers (the 60 and 79 kd peaks), and CA125, improvedsensitivity by more than 10% over that of CA125 alone. More recently,Zhang et al. (2004) identified three biomarkers for the detection ofearly stage ovarian cancer from serum proteomics analysis using multiplechips. They identified a 28 kDa band to be ApoAI (down-regulated), a12.8 kDa band to be a truncated form of TTR (down-regulated), and a 3.2kDa band as a cleavage fragment of inter-alpha-trypsin inhibitor heavychain H4 (up-regulated). Statistical analysis demonstrated thatcombining the three biomarkers (3.2, 12.8 and 28 kDa peaks) and CA125level, improved sensitivity by 9% over that of CA125 level alone.However, it should be noted that our results from multivariate analyseswere performed using values derived entirely from ELISA, a potentiallymore clinically relevant assay, rather than SELDI-TOF-MS intensityvalues.

One of the markers identified in the present study, ApoAI, is the majorapolipoprotein of high density lipoprotein and is an abundant plasmaprotein. Recently, Zhang et al. reported that ApoAI is differentiallyexpressed in patients with ovarian cancer and is a good marker for earlystage ovarian cancer. ApoAI levels have been found to also decreaseduring tangier disease and arteriosclerosis. Another marker identifiedin our study, TTR, is a secreted protein with a molecular mass of 13.8kDa that functions as a binding protein to transport thyroxine andretinal (vitamin A). TTR is decreased in patients with ovariancarcinoma, advanced cervical and endometrial carcinomas. Mahlck andGrankvist, showed that TTR concentrations are lower in women withcarcinoma of the ovary than in postmenopausal controls and the levelscorrelate inversely to tumor volume, suggesting prognostic significance.TTR is also known to decrease during severe liver disease, malnutritionand acute inflammation.

When analyzed as a marker by itself, Hb turned out to be a significantbiomarker for the detection of ovarian tumors (LMP), however, the markerwas not significantly differentially expressed in early stage malignanttumors with nonparametric analysis (although is was significant withparametric analysis). We were initially concerned with this findingsince mechanical handling and/or sample preparation can potentiallyresult in RBC lysis and Hb release. Therefore, to reduce the analysis ofHb released as an artifact, we omitted samples that were obviously redfrom our analyses and in subsequent careful repetitions of theseexperiments we confirmed that Hb is not an artifact but rather aspecific and significant serum biomarker of ovarian cancer. Alpha- andbeta-Hb chains are primarily involved in oxygen transport, forming aheterotetramer of two alpha chains and two beta chains in adulthemoglobin A. The alpha- and beta-Hb are proteins with molecular masses15.1 kDa and 15.8 kDa, respectively and have not been identified asmarkers for ovarian cancer previously. It has been reported thatbiochemical modifications of the erythrocyte membranes in women withovarian cancer may increase susceptibility to hemolysis of red bloodcells. Hb has been reported to also increase in polycythemia vera.

Transferrin is an iron binding transport protein, which can bind twoatoms of ferric iron in association with the binding of an anion,usually bicarbonate. Transferrin is responsible for the transport ofiron from sites of absorption and heme degradation to those of storageand utilization. The TF gene encodes a 77-80 kDa protein and has beenreported to decrease in the serum of patients with ovarian cancer. TFalso decreases during inflammation, nephrosis and haemochromatosis.

Recently, there has been strong criticism that SELDI-based analysesmostly identify highly abundant and acute phase proteins. Of the fivemarkers we identified and characterized in this study, only two markersfall in the class of acute phase proteins (TTR and TF). Moreover,preliminary analyses show that most of the remaining nine markers fromour original panels are not abundant serum proteins.

Statistical analysis of the ELISA data for LMP and malignant tumorsshowed all markers to be significant (p≦0.05) for LMP tumors, and allmarkers (with the exception of Hb [p=0.092]), were significant for earlystage malignant tumors (FIG. 8A-E). Multivariate analysis of thecombined markers improved detection of LMP tumors over CA125 by 18%, and30% for the mucinous subtype, while the combined markers, with CA125,did not significantly improve the ROC for all histological groups or themucinous subgroup (0.953 to 0.949, 0.926 to 0.932, respectively). Forthe malignant tumors, the statistical analysis resulted in an increaseof ROC (0.875 to 0.920) when comparing CA125 alone to our markers, whilethe addition of CA125 to our combined markers increased the ROC from0.920 to 0.971. The high sensitivity, specificity and ROC values for themucinous malignant tumors may be due to the low number of samplesanalyzed (n=3).

LMP (n=6) and malignant ovarian tumors (n=3) showed that our combinedmarkers improve detection of early stage tumors over CA125 by 8%, andimproved detection of the mucinous subgroup by 31%. The addition ofCA125 to our combined markers increased the ROC for early stagedetection of ovarian tumors for all histological groups (0.933 to0.959), but not for the mucinous group (0.959 to 0.955). Multivariateanalysis of early stage mucinous tumors (LMP and malignant) showed ourmarkers to greatly improve the detection of disease (ROC 0.959) ascompared to CA125 only (ROC 0.613). The addition of CA125 to themultivariate analysis of our markers did not seem to further improve thedetection of mucinous tumors (ROC 0.955).

Although our markers are differentially expressed in other diseases, thedifference in directionality (increasing or decreasing) and the abilityto combine multiple markers allows us to specifically detect early stageovarian cancer. In preliminary data, we have found our markers to beovarian cancer specific when comparing differential expression of ourmarkers in other cancers such as breast, colon, and epithelial, and toother diseases such as atherosclerosis. We plan to analyze our markersin additional cancers and diseases, as well as analyze additionalsamples. Thus, our characterized markers, even if they are not releaseddirectly by the tumor, could be used in combination with other markers,such as CA125, to improve the sensitivity and specificity of early stageovarian cancer.

In conclusion, we have characterized five m/z SELDI ovarian cancerbiomarker peaks and confirmed their differential expression in serumusing Western and ELISA assays. We have identified and characterizedTTR, Hb, ApoAI and TF as proteins that are differentially expressed inearly stage ovarian tumors. Together, these markers have improveddetection of early stage ovarian tumors relative to CA125 alone. Thesemarkers should facilitate the development of additional clinical assays,such as ELISAs, to improve early detection of ovarian cancer.

Example 3 Identification of Additional Ovarian Cancer Serum Biomarkers

This example demonstrates the identification of additional biomarkersfrom serum samples that exhibit sensitivity and specificity in detectingovarian neoplasia. These biomarkers were identified using SELDI asdescribed above. The following table lists the protein identity, m/zratios (in Daltons, “Marker”), cut point, sensitivity (“Sens”),specificity (“Spec”), accuracy (“Acc”) of each biomarker. Subsequentcolumns indicate the mean level observed for each biomarker in thescreening (normal and neoplasm) and validation (nonmalignant andmalignant) panels. N=140. Mean Level of Biomarker Validation CutScreening Non- Protein Marker Point Sens Spec Ace Normal NeoplasmMalignant Malignant M1953 1.18 0.70 0.63 0.67 1.57 4.25 2.95 3.67 M20651.70 0.63 0.70 0.66 1.73 3.23 2.63 2.81 M2216 0.87 0.65 0.63 0.64 1.042.60 1.81 2.30 M2928 1.35 0.55 0.83 0.69 0.79 2.06 1.38 1.84 M2937 1.790.69 0.72 0.70 1.81 3.35 2.14 3.36 M3143 1.59 0.65 0.65 0.65 1.64 2.592.02 2.47 M3423 0.48 0.57 0.74 0.66 0.57 1.54 0.71 1.59 M3427 0.58 0.660.67 0.67 0.63 1.66 1.04 1.52 M4144 4.26 0.74 0.63 0.69 4.46 6.79 5.256.59 M4375 0.98 0.76 0.59 0.67 0.94 1.54 0.97 1.61 M4456 2.01 0.60 0.870.73 1.36 3.05 1.54 3.19 M4629 3.48 0.35 0.93 0.64 2.22 3.20 2.42 3.21M5064 1.21 0.73 0.70 0.71 1.16 2.11 1.18 2.24 M6884 7.77 0.67 0.93 0.8010.46 6.87 10.12 6.54 M6931 10.08 0.70 0.80 0.75 12.29 8.31 12.05 7.85M7550 0.96 0.86 0.65 0.76 1.09 3.13 1.70 3.01 M7657 1.19 0.57 0.89 0.730.75 1.45 0.92 1.44 M7756 1.12 0.48 0.91 0.70 0.65 1.25 0.68 1.32 M81172.16 0.76 0.76 0.76 1.82 2.61 1.94 2.65 M10874 0.35 0.85 0.43 0.64 0.420.50 0.41 0.52 M12785 1.17 0.61 0.80 0.71 1.46 1.12 1.33 1.16 TTR M1379722.73 0.74 0.91 0.83 27.77 18.72 26.53 18.17 HBA M15074 1.38 0.81 0.760.78 1.50 4.47 2.23 4.42 HBB M15850 1.29 0.67 0.83 0.75 1.08 4.09 1.834.02 M16850 0.24 0.59 0.91 0.75 0.12 0.33 0.15 0.35 M18559 0.29 0.600.72 0.66 0.27 0.45 0.25 0.49 M18912 0.07 0.63 0.76 0.69 0.05 0.13 0.060.13 M18980 0.08 0.49 0.78 0.64 0.05 0.10 0.05 0.10 M19186 0.10 0.331.00 0.66 0.03 0.07 0.03 0.08 M20989 0.66 0.66 0.85 0.75 0.77 0.63 0.740.63 M22959 1.15 0.70 0.85 0.77 1.06 1.39 1.08 1.43 M27595 0.50 0.730.89 0.81 0.91 0.41 0.83 0.39 APOA1 M27977 0.54 0.48 0.85 0.66 1.52 0.791.47 0.71 M29190 0.81 0.38 0.89 0.64 0.69 0.76 0.72 0.75 M29512 0.680.68 0.67 0.68 0.61 0.76 0.65 0.75 M30103 0.56 0.84 0.52 0.68 0.55 0.750.57 0.76 M33217 12.80 0.49 0.83 0.66 11.61 12.84 11.92 12.81 M362960.55 0.59 0.85 0.72 0.42 0.76 0.42 0.82 M40067 0.30 0.60 0.93 0.77 0.470.29 0.47 0.27 M42401 0.36 0.60 0.70 0.65 0.34 0.40 0.33 0.42 α1-ATM53110 0.11 0.43 0.98 0.70 0.04 0.17 0.05 0.18 M53531 0.04 0.62 0.800.71 0.03 0.12 0.03 0.13 M54605 0.18 0.76 0.61 0.68 0.20 0.16 0.16 0.18TF M78715 1.05 0.78 0.85 0.81 1.46 0.78 1.38 0.73 M79909 1.28 0.63 0.870.75 1.71 1.13 1.69 1.05 M83689 0.04 0.63 0.72 0.67 0.03 0.10 0.05 0.10M84133 0.02 0.69 0.74 0.72 0.02 0.05 0.03 0.06 M90834 0.18 0.52 0.800.66 0.22 0.18 0.21 0.17 M91878 0.19 0.48 0.89 0.69 0.25 0.19 0.25 0.19M92935 0.24 0.59 0.89 0.74 0.29 0.21 0.29 0.20 M105778 0.06 0.65 0.800.73 0.09 0.05 0.09 0.04 IgG M106624 0.09 0.56 0.91 0.74 0.12 0.08 0.120.08

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method of screening for ovarian neoplasia in a subject comprising:(a) measuring hemoglobin in a tissue sample of the subject, and (b)comparing the measured hemoglobin of the tissue sample to a measurementof hemoglobin in normal tissue, wherein a two-fold or greater increasein the measured of hemoglobin of the tissue sample compared to themeasurement of hemoglobin in normal tissue is indicative of ovarianneoplasia.
 2. The method of claim 1, wherein the measuring furthercomprises measuring at least two biomarkers in the tissue sample,wherein the at least two biomarkers are selected from a first group ofbiomarkers whose up-regulation is associated with ovarian cancer and/ora second group of biomarkers whose down-regulation is associated withovarian cancer, wherein the first group of biomarkers consists of: (i) aprotein having an m/z of 1.953 kDa, 2.065 kDa, 2.216 kDa, 2.928 kDa,2.937 kDa, 3.143 kDa, 3.423 kDa, 3.427 kDa, 4.144 kDa, 4.375 kDa, 4.456kDa, 4.629 kDa, 5.064 kDa, 7.550 kDa, 7.657 kDa, 7.756 kDa, 8.117 kDa,10.874 kDa, 16.850 kDa, 18.559 kDa, 18.912 kDa, 18.98 kDa, 19.186 kDa,22.959 kDa, 29.19 kDa, 29.512 kDa, 30.103 kDa, 33.217 kDa, 36.296 kDa,42.401 kDa, 53.11 kDa (α1-AT), 53.531 kDa, 83.689 kDa, or 84.133 kDa;and wherein the second group of biomarkers consists of: (ii) a proteinhaving an m/z of 6.884 kDa, 6.931 kDa, 12.785 kDa (transthyretin),13.797 kDa (transthyretin), 20.989 kDa, 27.595 kDa, 27.977 kDa(apolipoprotein Al), 40.067 kDa, 54.605 kDa, 78.9 kDa (transferrin),79.909 kDa, 90.834 kDa, 91.878 kDa, 92.935 kDa, 105.778 kDa, or 106.624kDa (IgG); and comparing the measurements of the at least two biomarkersin the tissue sample to a known profile of the at least two biomarkersin normal tissue, wherein a measurement indicating a two-fold or greaterincrease in a member of the first group of biomarkers, or a two-fold orgreater decrease in a member of the second group of biomarkers, relativeto normal tissue, is indicative of ovarian neoplasia.
 3. The method ofclaim 2, wherein the at least two biomarkers comprise transthyretin(12.785 kDa and/or 13.797 kDa), apolipoprotein Al (27.977 kDa) and/ortransferrin (78.9 kDa) of the second group of biomarkers.
 4. The methodof claim 1, wherein the tissue sample comprises serum.
 5. The method ofclaim 1, wherein the measuring comprises spectrometry.
 6. The method ofclaim 5, wherein the spectrometry comprises surface enhanced laserdesorption/ionization (SELDI) mass spectrometry.
 7. The method of claim1, wherein the measuring comprises immunoassay.
 8. The method of claim1, wherein the first group of biomarkers consists of proteins having anm/z of 4.144 kDa, 4.456 kDa, 7.756 kDa, 15.074 kDa, 15.85 kDa, 18.912kDa, 22.959 kDa, 30.103 kDa and 53.531 kDa, and the second group ofbiomarkers consists of a protein having an m/z of 12.785 kDa.
 9. Themethod of claim 8, wherein the measuring comprises measuring each memberof the first and second groups of biomarkers.
 10. The method of claim 1,wherein the first group of biomarkers consists of proteins having an m/zof 3.143 kDa, 4.456 kDa, 5.064 kDa, 7.756 kDa, 8.117 kDa, 16.85 kDa, and18.559 kDa, and the second group of biomarkers consists of proteinshaving an m/z of 13.797 kDa, 20.989 kDa, 27.977 kDa, 78.715 kDa, 92.935kDa and 106.624 kDa.
 11. The method of claim 10, wherein the measuringcomprises measuring each member of the first and second groups ofbiomarkers.
 12. The method of claim 1, wherein the first group ofbiomarkers consists of proteins having an m/z of 4.456 kDa, 15.85 kDa,18.912 kDa, 22.959 kDa and 30.103 kDa.
 13. The method of claim 12,wherein the measuring comprises measuring each member of the first groupof biomarkers.
 14. The method of claim 13, further comprising: (a)measuring a validation biomarker panel I (VBPI) consisting of a firstVBPI group of biomarkers that consists of a protein having an m/z of3.143 kDa, and a second VBPI group of biomarkers that consists ofproteins having an m/z of 13.797 kDa, 20.989 kDa, 78.715 kDa and 106.624kDa; (b) wherein a measurement indicating a two-fold or greater increasein a member of the first VBPI group of biomarkers, or a two-fold orgreater decrease in a member of the second VBPI group of biomarkers,relative to normal tissue, is indicative of malignant ovarian neoplasia.15. The method of claim 14, further comprising: (a) measuring avalidation biomarker panel II (VBPII) consisting of a first VBPII groupof biomarkers that consists of proteins having an m/z of 5.064 kDa and16.85 kDa, and a second VBPII group of biomarkers that consists ofproteins having an m/z of 27.977 kDa and 92.935 kDa; (b) wherein ameasurement indicating a two-fold or greater increase in a member of thefirst VBPII group of biomarkers, or a two-fold or greater decrease in amember of the second VBPII group of biomarkers, relative to normaltissue, is indicative of malignant ovarian neoplasia.
 16. A method ofdetecting ovarian neoplasia in a test subject comprising: (a) measuringbiomarkers consisting of transthyretin, hemoglobin, ApoAI andtransferrin in a tissue sample from the test subject; and (b) comparingthe amount of the biomarkers in the tissue sample with the amount ofbiomarkers observed in a tissue sample from a normal subject, whereinincreased hemoglobin and decreased transthyretin, ApoAI and transferrinare indicative of ovarian neoplasia in the test subject.
 17. The methodof claim 16, wherein the ovarian neoplasia comprises a mucinous tumor.18. A method of screening for ovarian neoplasia in a subject comprising:(a) measuring at least three biomarkers in a tissue sample of thesubject, wherein the at least three biomarkers are selected from a firstgroup of biomarkers whose up-regulation is associated with ovariancancer and a second group of biomarkers whose down-regulation isassociated with ovarian cancer, wherein the first group of biomarkersconsists of: (i) a protein having an m/z of 1.953 kDa, 2.065 kDa, 2.216kDa, 2.928 kDa, 2.937 kDa, 3.143 kDa, 3.423 kDa, 3.427 kDa, 4.144 kDa,4.456 kDa, 4.629 kDa, 5.064 kDa, 7.550 kDa, 7.657 kDa, 7.756 kDa, 8.117kDa, 10.874 kDa, 15.074 kDa (hemoglobin A), 15.850 kDa (hemoglobin B),16.850 kDa, 18.559 kDa, 18.912 kDa, 18.98 kDa, 19.186 kDa, 22.959 kDa,29.19 kDa, 29.512 kDa, 33.217 kDa, 36.296 kDa, 42.401 kDa, 53.11 kDa(α1-AT), 53.531 kDa, 83.689 kDa, or 84.133 kDa; and wherein the secondgroup of biomarkers consists of: (ii) a protein having an m/z of 6.884kDa, 6.931 kDa, 20.989 kDa, 27.595 kDa, 40.067 kDa, 54.605 kDa, 79.909kDa, 90.834 kDa, 91.878 kDa, 92.935 kDa, 105.778 kDa, or 106.624 kDa(IgG); and (b) comparing the measurements of the at least threebiomarkers in the tissue sample to a known profile of the at least threebiomarkers in normal tissue, wherein a measurement indicating a two-foldor greater increase in a member of the first group of biomarkers, or atwo-fold or greater decrease in a member of the second group ofbiomarkers, relative to normal tissue, is indicative of ovarianneoplasia.
 19. The method of claim 18, wherein the measuring furthercomprises measuring at least one additional biomarker selected from:proteins having an m/z of 4.375 kDa and 30.103 kDa of the first group ofbiomarkers; and proteins having an m/z of 12.785 kDa (transthyretin),13.797 kDa (transthyretin), 27.977 kDa (ApoA1) and 78.715 kDa(transferrin) of the second group of biomarkers.
 20. The method of claim1, further comprising measuring CA125.
 21. A kit comprising: (a) atleast one agent that binds a biomarker selected from α-hemoglobin,β-hemoglobin, alpha1-antitrypsin (α1-AT) and any combination thereof;and (b) instructions for use of the at least one agent for determiningstatus of ovarian neoplasia in a test sample.
 22. The kit of claim 21,further comprising a container for housing the at least one agent. 23.The kit of claim 21, further comprising at least one agent that binds abiomarker selected from transthyretin, ApoAI, transferrin, CA125 and anycombination thereof.
 24. The kit of claim 21, further comprising asubstrate to which the at least one agent is bound.
 25. The kit of claim21, wherein the agent is an antibody that specifically binds thebiomarker.
 26. The kit of claim 21, wherein the agent is a massspectrometry probe.
 27. The kit of claim 21, wherein the status ofovarian neoplasia comprises absence of neoplasia, benign, low malignantpotential (LMP), malignant neoplasia.